Targeting pax2 for the treatment of breast cancer

ABSTRACT

The present application provides methods of prevention and/or treatment of breast cancer in a subject by inhibiting expression of PAX2. In the cancer treatment methods disclosed, the method of inhibiting expression of PAX2 can be by administration of a nucleic acid encoding an siRNA for PAX2. A method of treating cancer in a subject by administering DEFB1 is also provided. Similarly, provided is a method of treating cancer in a subject by increasing expression of DEFB1 in the subject.

The present application is a continuation application of U.S. patentapplication Ser. No. 13/116,752, filed on May 26, 2011, which is acontinuation of U.S. patent application Ser. No. 12/708,294, filed onFeb. 18, 2010, now U.S. Pat. No. 8,080,534, which is acontinuation-in-part application of U.S. patent application Ser. No.12/090,191, filed Sep. 15, 2008, now U.S. Pat. No. 7,964,577, as anational stage application of PCT/US2006/040215 filed Oct. 16, 2006,which claims priority to U.S. Patent Application No. 60/726,921, filedOct. 14, 2005. The entirety of all of the aforementioned applications isincorporated herein by reference.

BACKGROUND

Breast cancer is the most common cause of cancer in women and the secondmost common cause of cancer death in women in the U.S. While themajority of new breast cancers are diagnosed as a result of anabnormality seen on a mammogram, a lump or change in consistency of thebreast tissue can also be a warning sign of the disease. Heightenedawareness of breast cancer risk in the past decades has led to anincrease in the number of women undergoing mammography for screening,leading to detection of cancers in earlier stages and a resultantimprovement in survival rates. Still, breast cancer is the most commoncause of death in women between the ages of 45 and 55.

It is known that many types of cancer are caused by genetic aberrations,i.e., mutations. The accumulation of mutations and the loss of cellularcontrol functions cause progressive phenotypic changes from normalhistology to early pre-cancer such as intraepithelial neoplasia (IEN) toincreasingly severe IEN to superficial cancer and finally to invasivedisease. Although this process can be relatively aggressive in somecases, it generally occurs relatively slowly over years and evendecades. Oncogene addiction is the physiologic dependence of cancercells on the continued activation or overexpression of single oncogenesfor maintaining the malignant phenotype. This dependence occurs in themilieu of the other changes that mark neoplastic progression.

The long period of progression to invasive cancer provides anopportunity for clinical intervention. Therefore, it is important toidentify biomarkers that are indicative of pre-cancerous conditions sothat treatment measures can be taken to prevent or delay the developmentof invasive cancer.

SUMMARY

One aspect of the present invention relates to a method for preventingor treating a breast condition in a subject. The method comprisesadministering to a breast tissue of the subject, a composition thatinhibits PAX2 expression or PAX2 activity.

In one embodiment, the breast condition is breast cancer or mammaryintraepithelial neoplasia (MIN).

In another embodiment, the inhibiting expression of PAX2 comprisesadministering to the breast cancer tissue or MIN tissue in the subject anucleic acid encoding an siRNA for PAX2.

In a related embodiment, the siRNA comprises a sequence selected fromthe group consisting of SEQ ID NOS: 3-6 and 11-15.

In another embodiment, the composition comprises an oligonucleotide thatinhibits PAX2 binding to the DEFB1 promoter.

In a related embodiment, the oligonucleotide comprises SEQ ID NO:1 inforward or reverse orientation.

In a related embodiment, the oligonucleotide comprises the sequence ofX1GGAACX2, wherein X1 and X2 are 0 to 30 nucleotides complementary tonucleotides contiguous to SEQ ID NO:1 in the DEFB1 coding sequence.

In a related embodiment, the oligonucleotide comprises a sequenceselected from the group consisting of SEQ ID NOS: 18-21, 25, 26, 28 and29.

In another embodiment, the composition comprises a blocker of RASsignaling pathway.

In another embodiment, the composition comprises an antagonist selectedfrom the group consisting of antagonists of angiotensin II, antagonistsof angiotensin II receptor, antagonists of angiotensin-converting enzyme(ACE), antagonists of mitogen-activated protein kinase (MEK),antagonists of (extracellular signal-regulated kinase) ERK1,2, andantagonists of signal transducer and activator of transcription 3(STAT3).

Also disclosed is a method of treating breast cancer or MIN in asubject, comprising enhancing expression of DEFB1 in a breast cancertissue or MIN tissue in the subject.

In one embodiment, the enhancing expression of DEFB1 comprisesadministering to the breast cancer tissue or MIN tissue in the subjectan efficient amount of DEFB1.

In another embodiment, the enhancing expression of DEFB1 comprisesadministering to the breast cancer tissue or MIN tissue in the subjectan efficient amount of an expression vector encoding DEFB1.

Also disclosed is a method for treating a breast condition in a subject,comprising: (a) determining the PAX2-to-DEFB1 expression ratio in adiseased breast tissue from said subject; (b) determining the ER/PRstatus of said diseased breast tissue from said subject; and (c) basedon the result of (a) and (b), administering to a breast tissue of saidsubject, a composition that (1) inhibits PAX2 expression or PAX2activity, (2) expresses DEFB1 or (3) inhibits PAX2 expression or PAX2activity and expresses DEFB1.

BRIEF DESCRIPTION OF THE DRAWINGS

The accompanying drawings illustrate one or more embodiments of theinvention and, together with the written description, serve to explainthe principles of the invention. Wherever possible, the same referencenumbers are used throughout the drawings to refer to the same or likeelements of an embodiment.

FIGS. 1A-1D show quantitative RT-PCR (QRT-PCR) analysis ofbeta-defensin-1 (DEFB1) expression.

FIG. 2 shows microscopic analysis of DEFB1 induced changes in membraneintegrity and cell morphology. Membrane ruffling is indicated by blackarrows and apoptotic bodies are indicated white arrows.

FIG. 3 shows analysis of DEFB1 Cytotoxicity in Prostate Cancer Cells.The prostate cell lines DU145, PC3 and LNCaP were treated with PonA toinduce DEFB1 expression for 1-3 days after which MTT assay was performedto determine cell viability. Results represent mean±s.d., n=9.

FIGS. 4A and 4B show induction of cell death in DU145 and PC3 cells byDEFB1.

FIGS. 5A-5L show pan-caspase analysis following DEFB1 induction.

FIG. 6 show silencing of paired box homeotic gene 2 (PAX2) proteinexpression following PAX2 siRNA treatment.

FIG. 7 shows analysis of prostate cancer cells growth after treatmentwith PAX2 siRNA.

FIG. 8 shows analysis of cell death following siRNA silencing of PAX2.Results represent mean±s.d., n=9.

FIG. 9 shows analysis of caspase activity.

FIGS. 10A-10C show analysis of apoptotic factors following PAX2 siRNAtreatment.

FIG. 11 shows model of PAX2 binding to DNA recognition sequence.

FIG. 12 illustrates the DEFB1 reporter construct.

FIG. 13 shows inhibition of PAX2 results in DEFB1 Expression.

FIG. 14 shows that inhibition of PAX2 results in increased DEFB1promoter activity.

FIG. 15 shows that DEFB1 expression causes loss of membrane integrity.

FIG. 16 shows that PAX2 inhibition results in loss of membraneintegrity.

FIGS. 17A and 17B show ChIP analysis of PAX2 binding to DEFB1 promoter.In FIG. 17A, Lane 1 contains a 100 bp molecular weight marker. Lane 2 isa positive control representing 160 bp region of the DEFB1 promoteramplified from DU145 before cross-linking and immunoprecipitation. Lane3 is a negative control representing PCR performed without DNA. Lanes 4and 5 are negative controls representing PCR from immunoprecipitationsperformed with IgG from cross-linked DU145 and PC3, respectively. PCRamplification of 25 pg of DNA (lane 6 and 8) and 50 pg of DNA (lane 7and 9) immunoprecitipated with anti-PAX2 antibody after crosslinkingshow 160 bp promoter fragment in DU145 and PC3, respectively. In FIG.17B, Lane 1 contains a 100 bp molecular weight marker. Lane 2 is apositive control representing 160 bp region of the DEFB1 promoteramplified from DU145 before cross-linking and immunoprecipitation. Lane3 is a negative control representing PCR performed without DNA. Lane 4and 5 are negative controls representing PCR from immunoprecipitationsperformed with IgG from cross-linked DU145 and PC3, respectively. PCRamplification of 25 pg of DNA (lane 6 and 8) and 50 pg of DNA (lane 7and 9) immunoprecitipated with anti-PAX2 antibody after crosslinkingshow 160 bp promoter fragment in DU145 and PC3, respectively

FIG. 18 shows predicted structure of the PrdPD and PrdHD with DNA.

FIG. 19 shows comparison of consensus sequences of different paireddomains. At the top of the Figure is drawn a schematic representation ofprotein±DNA contacts described in the crystallographic analysis of thePrd-paired-domain±DNA complex. Empty boxes indicate a-helices, shadedboxes indicates b-sheets and a thick line indicate a b-turn. Contactingamino acids are shown by single-letter code. Only direct amino acid±basecontacts are shown. Empty circles indicate major groove contacts whilered arrows indicate minor groove contacts. This scheme is aligned to allknown consensus sequences for paired-domain proteins (top strands onlyare shown). Vertical lines between consensus sequences indicateconserved base-pairs. Numbering of the positions is shown at the bottomof the Figure.

FIG. 20 shows targeting PAX2 as a chemopreventive strategy.

FIG. 21 shows effect of angiotensin II (Ang II) on PAX2 expression inDU145 Cells.

FIGS. 22A-22B show effect of Losartan (Los) on PAX2 expression in DU145.

FIG. 23 shows Los blocks AngII effect on PAX2 expression in DU145.

FIG. 24 shows AngII increases DU145 cell proliferation.

FIGS. 25A, 25B and 25C show effect of Los and MAP Kinase inhibitors onPAX2 expression in DU145 cells. FIG. 25A shows treatment of DU145 cellswith Losartan suppresses phosphor-ERK 1/2 and PAX2 expression; FIG. 25Bshows MEK kinase inhibitors and AICAR suppresses PAX2 proteinexpression; FIG. 25C shows MEK kinase inhibitors and Losartan suppressesphospho-STAT3 protein expression.

FIGS. 26A and 26B show effect of Los and MEK kinase inhibitors on PAX2activation in DU145 cells

FIG. 27 shows AngII increases PAX2 and decreases DEFB1 expression inhPrEC cells.

FIG. 28 shows schematic of AngII signaling and PAX2 prostate cancer.

FIG. 29 shows schematic of blocking PAX2 expression as a therapy forprostate cancer.

FIG. 30 shows comparison of DEFB1 and PAX2 expression with GleasonScore.

FIGS. 31A and 31B show PAX2-DEFB1 ratio as a predictive factor forprostate cancer development.

FIG. 32 shows the Donald Predictive Factor (DPF) is based on therelative PAX2-DEFB1 expression ratio.

FIGS. 33A and 33B show analysis of hBD-1 expression in human prostatetissue.

FIGS. 34A and 34B show analysis of hBD-1 expression in prostate celllines. FIG. 34A shows hBD-1 expression levels compared relative to hPrECcells in prostate cancer cell lines before and after hBD-1 induction. Anasterisk represents statistically higher expression levels compared tohPrEC. Double asterisks represent statistically significant levels ofexpression compared to the cell line before hBD-1 induction (Student'st-test, p<0.05). FIG. 34B shows ectopic hBD-1 expression verified in theprostate cancer cell line DU145 by immunocytochemistry. hPrEC cells werestained for hBD-1 as appositive control (A: DIC and B: fluorescence).DU145 cells were transfected with hBD-1 and induced for 18 hours (C: DICand D: fluorescence). Sizebar=20 μM.

FIG. 35 shows analysis of hBD-1 cytotoxicity in prostate cancer cells.Each bar represents the mean±S.E.M. of three independent experimentsperformed in triplicate.

FIGS. 36A and 36B show QRT-PCR analysis of hBD-1 and cMYC expression inLCM human prostate tissue sections of normal, PIN and tumor. Expressionfor each gene is presented as expression ratios compared to β-actin.FIG. 36A shows comparison of hBD-1 expression levels in normal, PIN andtumor sections. FIG. 36B shows comparison of cMYC expression level innormal, PIN and tumor sections.

FIG. 37 shows QRT-PCR analysis of hBD1 expression following PAX2knockdown with siRNA. hBD-1 expression levels are presented asexpression ratios compared to β-actin. An asterisk representsstatistically higher expression levels compared to the cell line beforePAX2 siRNA treatment (Student's t-test, p<0.05).

FIGS. 38A and 38B show silencing of PAX2 protein expression followingPAX2 siRNA treatment. FIG. 38A shows PAX2 expression examined by Westernblot analysis in HPrEC prostate primary cells (lane 1) and in DU145(lane 2), PC3 (lane 3) and LNCaP (lane 4) prostate cancer cells. Blotswere stripped and re-probed for -actin as an internal control to ensureequal loading. FIG. 38B shows Western blot analysis of DU145, PC3 andLNCaP all confirmed knockdown of PAX2 expression following transfectionwith PAX2 siRNA duplex. Again, blots were stripped and re-probed forβ-actin as an internal control.

FIG. 39 shows analysis of prostate cancer cells growth after treatmentwith PAX2 siRNA. Bar=20 μm.

FIG. 40 shows analysis of cell death following siRNA silencing of PAX2.Results represent mean±SD, n=9.

FIG. 41 shows analysis of caspase activity. Bar=20 μm.

FIGS. 42A-42C show analysis of apoptotic factors following PAX2 siRNAtreatment. Results represent mean±SD, n=9. Asterisks representsstatistical differences (p<0.05).

DETAILED DESCRIPTION

One aspect of the present invention provides a method of preventing ortreating breast cancer in a subject. The method includes administeringto the subject a composition comprising an inhibitor of PAX2 expressionor PAX2 activity, or an enhancer of DEFB-1 expression or DEFB-1activity. In one embodiment, the subject is diagnosed with mammaryintraepithelial neoplasia (MIN).

In some aspects, PAX2 is upregulated in breast tissue prior to MIN.Thus, also provided is a method of treating or preventing MIN in asubject. The method comprises administering to the subject a compositioncomprising an inhibitor of PAX2 expression or PAX2 activity, or anenhancer of DEFB-1 expression or DEFB-1 activity.

“Activities” of a protein include, for example, transcription,translation, intracellular translocation, secretion, phosphorylation bykinases, cleavage by proteases, homophilic and heterophilic binding toother proteins, ubiquitination. In some aspects, “PAX2 activity” refersspecifically to the binding of PAX2 to the DEFB-1 promoter.

Breast Cancer

The commonly used screening methods for breast cancer include self andclinical breast exams, x-ray mammography, and breast Magnetic ResonanceImaging (MRI). The most recent technology for breast cancer screening isultrasound computed tomography, which uses sound waves to create athree-dimensional image and detect breast cancer without the use ofdangerous radiation used in x-ray mammography. Genetic testing may alsobe used. Genetic testing for breast cancer typically involves testingfor mutations in the BRCA genes. It is not a generally recommendedtechnique except for those at elevated risk for breast cancer.

The incidence of breast cancer, a leading cause of death in women, hasbeen gradually increasing in the United States over the last thirtyyears. While the pathogenesis of breast cancer is unclear,transformation of normal breast epithelium to a malignant phenotype maybe the result of genetic factors, especially in women under 30. Thediscovery and characterization of BRCA1 and BRCA2 has recently expandedour knowledge of genetic factors which can contribute to familial breastcancer. Germ-line mutations within these two loci are associated with a50 to 85% lifetime risk of breast and/or ovarian cancer. However, it islikely that other, non-genetic factors also have a significant effect onthe etiology of the disease. Regardless of its origin, breast cancermorbidity and mortality increases significantly if it is not detectedearly in its progression. Thus, considerable effort has focused on theearly detection of cellular transformation and tumor formation in breasttissue.

Currently, the principal manner of identifying breast cancer is throughdetection of the presence of dense tumorous tissue. This may beaccomplished to varying degrees of effectiveness by direct examinationof the outside of the breast, or through mammography or other X-rayimaging methods. The latter approach is not without considerable cost,however. Every time a mammogram is taken, the patient incurs a smallrisk of having a breast tumor induced by the ionizing properties of theradiation used during the test. In addition, the process is expensiveand the subjective interpretations of a technician can lead toimprecision, e.g., one study showed major clinical disagreements forabout one-third of a set of mammograms that were interpretedindividually by a surveyed group of radiologists. Moreover, many womenfind that undergoing a mammogram is a painful experience. Accordingly,the National Cancer Institute has not recommended mammograms for womenunder fifty years of age, since this group is not as likely to developbreast cancers as are older women. It is compelling to note, however,that while only about 22% of breast cancers occur in women under fifty,data suggests that breast cancer is more aggressive in pre-menopausalwomen.

PAX2

PAX genes are a family of nine developmental control genes coding fornuclear transcription factors. They play an important role inembryogenesis and are expressed in a very ordered temporal and spatialpattern. They all contain a “paired box” region of 384 base pairsencoding a DNA binding domain which is highly conserved throughoutevolution (Stuart, E T, et al. 1994). The influence of Pax genes ondevelopmental processes has been demonstrated by the numerous naturalmouse and human syndromes that can be attributed directly to even aheterozygous insufficiency in a Pax gene. A PAX2 sequence is given inDressler, et al. 1990. The amino acid sequences of the human PAX2protein and its variants, as well as the DNA sequences encoding theproteins, are listed in SEQ ID NOS: 39-50 (SEQ ID NO:39, amino acidsequence encoded by exon 1 of the human PAX2 gene; SEQ ID NO:40, humanPAX2 gene promoter and exon 1; SEQ ID NO:41, amino acid sequence of thehuman PAX2; SEQ ID NO:42, human PAX2 gene; SEQ ID NO:43, amino acidsequence of the human PAX2 gene variant b; SEQ ID NO:44, human PAX2 genevariant b; SEQ ID NO:45, amino acid sequence of the human PAX2 genevariant c; SEQ ID NO:46, human PAX2 gene variant c; SEQ ID NO:47, aminoacid sequence of the human PAX2 gene variant d; SEQ ID NO:48, human PAX2gene variant d; SEQ ID NO:49, amino acid sequence of the human PAX2 genevariant e; SEQ ID NO:50 human PAX2 gene variant e). It has been reportedthat PAX2 suppresses DEFB-1 expression by binding to the DEFB-1 promoter(Bose S K et al., Mol Immunol 2009, 46:1140-8) at a 5′-CCTTG-3′ (SEQ IDNO:1) recognition site immediately adjacent to the DEFB1 TATA box. Insome references, the binding site is also referred to as the 3′-GTTCC-5′(SEQ ID NO:1) or 5′-CAAGG-3′ (SEQ ID NO:2) recognition site, which isthe sequence on the opposite strand. The two sequences both refer to thePAX2 binding site on the DEFB1 promoter. Examples of cancers in whichPAX2 expression has been detected are listed in Table 1

TABLE 1 PAX2-expressing cancers Estimated Estimated Estimated PAX2 NewDeaths New Estimated Expressing Cases in in Cases Deaths Cancers US USGlobal Global Prostate 234,460 27,350 679,023 221,002 Breast 214,60041,430 1,151,298 410,712 Ovarian 20,180 15,310 204,500 124,860 Renal38,890 12,840 208,479 101,895 Brain 12,820 18,820 189,485 141,650Cervical 9,710 3,700 493,243 273,505 Bladder 61,420 13,060 356,556145,009 Leukemia 35,020 22,280 300,522 222,506 Kaposi Data Not Data NotData Not Data Not Sarcoma Available Available Available AvailableTOTAL(approx.) 627,100 154,790 3,583,106 1,641,139

DEFB1

Beta-defensins are cationic peptides with broad-spectrum antimicrobialactivity that are products of epithelia and leukocytes. These two exon,single gene products are expressed at epithelial surfaces and secretedat sites including the skin, cornea, tongue, gingiva, salivary glands,esophagus, intestine, kidney, urogenital tract, and the respiratoryepithelium. To date, five beta-defensin genes of epithelial origin havebeen identified and characterized in humans: DEFB1 (Bensch et al.,1995), DEFB 2 (Harder et al., 1997), DEFB3 (Harder et al., 2001; Jia etal., 2001), DEFB4, and HE2/EP2. The amino acid sequence of human DEFB1and the human DEFB1 gene sequences are shown in SEQ ID NOS:63 and 64,respectively.

The primary structure of each beta-defensin gene product ischaracterized by small size, a six cysteine motif, high cationic chargeand exquisite diversity beyond these features. The most characteristicfeature of defensin proteins is their six-cysteine motif that forms anetwork of three disulfide bonds. The three disulfide bonds in thebeta-defensin proteins are between C1-05, C2-C4 and C3-C6. The mostcommon spacing between adjacent cysteine residues is 6, 4, 9, 6, 0. Thespacing between the cysteines in the beta-defensin proteins can vary byone or two amino acids except for C5 and C6, located nearest the carboxyterminus. In all known vertebrate beta-defensin genes, these twocysteine residues are adjacent to each other.

A second feature of the beta-defensin proteins is their small size. Eachbeta-defensin gene encodes a preproprotein that ranges in size from 59to 80 amino acids with an average size of 65 amino acids. This geneproduct is then cleaved by an unknown mechanism to create the maturepeptide that ranges in size from 36 to 47 amino acids with an averagesize of 45 amino acids. The exceptions to these ranges are the EP2IHE2gene products that contain the beta-defensin motif and are expressed inthe epididymis.

A third feature of beta-defensin proteins is the high concentration ofcationic residues. The number of positively charged residues (arginine,lysine, histidine) in the mature peptide ranges from 6 to 14 with anaverage of 9.

The final feature of the beta-defensin gene products is their diverseprimary structure but apparent conservation of tertiary structure.Beyond the six cysteines, no single amino acid at a given position isconserved in all known members of this protein family. However, thereare positions that are conserved that appear to be important forsecondary and tertiary structures and function.

Despite the great diversity of the primary amino acid sequence of thebeta-defensin proteins, the limited data suggests that the tertiarystructure of this protein family is conserved. The structural core is atriple-stranded, antiparallel beta-sheet, as exemplified for theproteins encoded by BNBD-12 and DEFB2. The three beta-strands areconnected by a beta-turn, and an alpha-hairpin loop, and the secondbeta-strand also contains a beta-bulge. When these structures are foldedinto their proper tertiary structure, the apparently random sequences ofcationic and hydrophobic residues are concentrated into two faces of aglobular protein. One face is hydrophilic and contains many of thepositively charged side chains and the other is hydrophobic. Insolution, the HBD-2 protein encoded by the DEFB2 gene exhibited analpha-helical segment near the N-terminus not previously ascribed tosolution structures of alpha-defensins or to the beta-defensin BNBD-12.The amino acids whose side chains are directed toward the surface of theprotein are less conserved between beta defensin proteins while theamino acid residues in the three beta-strands of the core beta-sheet aremore highly conserved.

Beta-defensin peptides are produced as pre-pro-peptides and then cleavedto release a C-terminal active peptide fragment; however the pathwaysfor the intracellular processing, storage and release of the humanbeta-defensin peptides in airway epithelia are unknown.

Inhibitors of PAX2 Expression or PAX2 Activity Functional Nucleic Acids

The inhibitor of the disclosed methods can be a functional nucleic acidthat inhibits PAX2 expression. Functional nucleic acids are nucleic acidmolecules that have a specific function, such as binding a targetmolecule or catalyzing a specific reaction. Functional nucleic acidmolecules can be divided into the following categories, which are notmeant to be limiting. For example, functional nucleic acids includeantisense molecules, aptamers, ribozymes, triplex forming molecules,RNAi, and external guide sequences. The functional nucleic acidmolecules can act as affectors, inhibitors, modulators, and stimulatorsof a specific activity possessed by a target molecule, or the functionalnucleic acid molecules can possess a de novo activity independent of anyother molecules.

Functional nucleic acid molecules can interact with any macromolecule,such as DNA, RNA, polypeptides, or carbohydrate chains. Thus, functionalnucleic acids can interact with the mRNA of PAX2 or the genomic DNA ofPAX2 or they can interact with the polypeptide PAX2. Often functionalnucleic acids are designed to interact with other nucleic acids based onsequence homology between the target molecule and the functional nucleicacid molecule. In other situations, the specific recognition between thefunctional nucleic acid molecule and the target molecule is not based onsequence homology between the functional nucleic acid molecule and thetarget molecule, but rather is based on the formation of tertiarystructure that allows specific recognition to take place.

Antisense molecules are designed to interact with a target nucleic acidmolecule through either canonical or non-canonical base pairing. Theinteraction of the antisense molecule and the target molecule isdesigned to promote the destruction of the target molecule through, forexample, RNAseH mediated RNA-DNA hybrid degradation. Alternatively theantisense molecule is designed to interrupt a processing function thatnormally would take place on the target molecule, such as transcriptionor replication. Antisense molecules can be designed based on thesequence of the target molecule. Numerous methods for optimization ofantisense efficiency by finding the most accessible regions of thetarget molecule exist. Exemplary methods would be in vitro selectionexperiments and DNA modification studies using DMS and DEPC. It ispreferred that antisense molecules bind the target molecule with adissociation constant (Kd) less than or equal to 10⁻⁶, 10⁻⁸, 10⁻¹⁰, or10⁻¹².

Aptamers are molecules that interact with a target molecule, preferablyin a specific way. Typically aptamers are small nucleic acids rangingfrom 15-50 bases in length that fold into defined secondary and tertiarystructures, such as stem-loops or G-quartets. Aptamers can bind smallmolecules, such as ATP and theophiline, as well as large molecules, suchas reverse transcriptase and thrombin. Aptamers can bind very tightlywith Kd's from the target molecule of less than 10⁻¹² M. It is preferredthat the aptamers bind the target molecule with a Kd less than 10⁻⁶,10⁻⁸, 10⁻¹⁰, or 10⁻¹². Aptamers can bind the target molecule with a veryhigh degree of specificity. For example, aptamers have been isolatedthat have greater than a 10,000 fold difference in binding affinitiesbetween the target molecule and another molecule that differ at only asingle position on the molecule. It is preferred that the aptamer have aKd with the target molecule at least 10, 100, 1000, 10,000, or 100,000fold lower than the Kd with a background binding molecule. It ispreferred when doing the comparison for a polypeptide for example, thatthe background molecule be a different polypeptide.

Ribozymes are nucleic acid molecules that are capable of catalyzing achemical reaction, either intramolecularly or intermolecularly.Ribozymes are thus catalytic nucleic acid. It is preferred that theribozymes catalyze intermolecular reactions. There are a number ofdifferent types of ribozymes that catalyze nuclease or nucleic acidpolymerase type reactions which are based on ribozymes found in naturalsystems, such as hammerhead ribozymes, hairpin ribozymes, andtetrahymena ribozymes. There are also a number of ribozymes that are notfound in natural systems, but which have been engineered to catalyzespecific reactions de novo. Preferred ribozymes cleave RNA or DNAsubstrates, and more preferably cleave RNA substrates. Ribozymestypically cleave nucleic acid substrates through recognition and bindingof the target substrate with subsequent cleavage. This recognition isoften based mostly on canonical or non-canonical base pair interactions.This property makes ribozymes particularly good candidates for targetspecific cleavage of nucleic acids because recognition of the targetsubstrate is based on the target substrates sequence.

Triplex forming functional nucleic acid molecules are molecules that caninteract with either double-stranded or single-stranded nucleic acid.When triplex molecules interact with a target region, a structure calleda triplex is formed, in which there are three strands of DNA forming acomplex dependant on both Watson-Crick and Hoogsteen base-pairing.Triplex molecules are preferred because they can bind target regionswith high affinity and specificity. It is preferred that the triplexforming molecules bind the target molecule with a Kd less than 10⁻⁶,10⁻⁸, 10⁴⁰, or 10⁴².

External guide sequences (EGSs) are molecules that bind a target nucleicacid molecule forming a complex, and this complex is recognized by RNaseP, which cleaves the target molecule. EGSs can be designed tospecifically target a RNA molecule of choice. RNAse P aids in processingtransfer RNA (tRNA) within a cell. Bacterial RNAse P can be recruited tocleave virtually any RNA sequence by using an EGS that causes the targetRNA:EGS complex to mimic the natural tRNA substrate. Similarly,eukaryotic EGS/RNAse P-directed cleavage of RNA can be utilized tocleave desired targets within eukaryotic cells.

Gene expression can also be effectively silenced in a highly specificmanner through RNA interference (RNAi). This silencing was originallyobserved with the addition of double stranded RNA (dsRNA). Once dsRNAenters a cell, it is cleaved by an RNase III-like enzyme, Dicer, intodouble stranded small interfering RNAs (siRNA) 21-23 nucleotides inlength that contains 2 nucleotide overhangs on the 3′ ends. In an ATPdependent step, the siRNAs become integrated into a multi-subunitprotein complex, commonly known as the RNAi induced silencing complex(RISC), which guides the siRNAs to the target RNA sequence. At somepoint the siRNA duplex unwinds, and it appears that the antisense strandremains bound to RISC and directs degradation of the complementary mRNAsequence by a combination of endo and exonucleases. However, the effectof iRNA or siRNA or their use is not limited to any type of mechanism.

Short Interfering RNA (siRNA) is a double-stranded RNA that can inducesequence-specific post-transcriptional gene silencing, therebydecreasing or even inhibiting gene expression. In one example, an siRNAtriggers the specific degradation of homologous RNA molecules, such asmRNAs, within the region of sequence identity between both the siRNA andthe target RNA. For example, WO 02/44321 discloses siRNAs capable ofsequence-specific degradation of target mRNAs when base-paired with 3′overhanging ends, herein incorporated by reference for the method ofmaking these siRNAs. Sequence specific gene silencing can be achieved inmammalian cells using synthetic, short double-stranded RNAs that mimicthe siRNAs produced by the enzyme dicer. siRNA can be chemically or invitro-synthesized or can be the result of short double-strandedhairpin-like RNAs (shRNAs) that are processed into siRNAs inside thecell. Synthetic siRNAs are generally designed using algorithms and aconventional DNA/RNA synthesizer. Suppliers include Ambion (Austin,Tex.), ChemGenes (Ashland, Mass.), Dharmacon (Lafayette, Colo.), GlenResearch (Sterling, Va.), MWB Biotech (Esbersberg, Germany), Proligo(Boulder, Colo.), and Qiagen (Vento, The Netherlands). siRNA can also besynthesized in vitro using kits such as Ambion's SILENCER® siRNAConstruction Kit. Disclosed herein are any siRNA designed as describedabove based on the sequences for PAX2.

The production of siRNA from a vector is more commonly done through thetranscription of a short hairpin RNAs (shRNAs). Kits for the productionof vectors comprising shRNA are available, such as, for example,Imgenex's GENESUPPRESSOR™ Construction Kits and Invitrogen's BLOCK-IT™inducible RNAi plasmid and lentivirus vectors. Disclosed herein are anyshRNA designed as described above based on the sequences for the hereindisclosed inflammatory mediators.

In certain embodiments, the functional nucleic acids include siRNAs thatinhibit expression of PAX 2 (anti-PAX2 siRNA). Examples of anti-PAX2siRNAs include, but are not limited to, siRNAs having the sequences of(5′ to 3′ direction):

(SEQ ID NO: 3) AUAGACUCGACUUGACUUCUU, (SEQ ID NO: 4)AUCUUCAUCACGUUUCCUCUU, (SEQ ID NO: 5) GUAUUCAGCAAUCUUGUCCUU,(SEQ ID NO: 6) GAUUUGAUGUGCUCUGAUGUU, (SEQ ID NO: 11)ACCCGACTATGTTCGCCTGG, (SEQ ID NO: 12) AAGCTCTGGATCGAGTCTTTG,(SEQ ID NO: 13) ATGTGTCAGGCACACAGACG, (SEQ ID NO: 14)GUCGAGUCUAUCUGCAUCCUU, (SEQ ID NO: 15) GGAUGCAGAUAGACUCGACUU,and fragments of at least 10 nucleic acids and conservative variantsthereof; and combinations thereof.

In other embodiments, the functional nucleic acids include antisense RNAto PAX2 and oligonuclotides that interfere with or inhibit the bindingof PAX2 to the DEFB1 promoter. The oligonucleotide can be complementaryto the sequence of PAX2 that binds to the DEFB1 promoter. Alternatively,the oligonucleotide can interact with the PAX2 in a way that inhibitsbinding to DEFB1. This interaction can be based on three-dimensionalstructure rather than primary nucleotide sequence.

PAX proteins are a family of transcription factors conserved duringevolution and able to bind specific DNA sequences through a domainscalled a “paired domain” and a “homeodomain”. The paired domain (PD) isa consensus sequence shared by certain PAX proteins (e.g., PAX2 andPAX6). The PD directs DNA binding of amino acids located in the a3-helixforming a DNA-Protein complex. For PAX2, the amino acids in the HDrecognize and interact specifically with a CCTTG (SEQ ID NO:1) DNA coresequence. Oligonucleotides include this sequence or its complement areexpected to be inhibitors. A critical DNA region in the DEFB1 promoterfor PAX2 protein binding has the sequence of AAGTTCACCCTTGACTGTG (SEQ IDNO: 16).

In one embodiment, the oligonucleotide has the sequence of V-CCTTG-W(SEQ ID NO: 17), wherein V and W are nucleotide sequences of 1 to 35nucleotides. In certain embodiments, V or W or both comprise contiguousnucleotide sequences that normally flank the PAX2 binding site of DEFB1promoter. Alternatively, the nucleotide sequences of V and/or W may beunrelated to the DEFB1 promoter, and selected randomly to avoidinterference with the PAX2 recognition sequence.

Other examples of oligonucleotides that inhibit PAX2 binding to theDEFB1 promoter include, but are not limited to, oligonucleotide havingthe sequences of (5′ to 3′ direction):

(SEQ ID NO: 18) CTCCCTTCAGTTCCGTCGAC, (SEQ ID NO: 19)CTCCCTTCACCTTGGTCGAC, (SEQ ID NO: 20)ACTGTGGCACCTCCCTTCAGTTCCGTCGACGAGGTTGTGC, and (SEQ ID NO: 21)ACTGTGGCACCTCCCTTCACCTTGGTCGACGAGGTTGTGC.

Other Inhibitors

Besides functional nucleotides, the inhibitors of PAX2 expression orPAX2 activity can be any small molecule that interferes or inhibitsbinding of PAX2 to the DEFB1 promoter. The inhibitors of PAX2 expressionor PAX2 activity can also be an antagonist of angiotensin II or anantagonist of angiotensin-converting enzyme (ACE). For example, theinhibitor can be enalapril or/and an antagonist of angiotensin II type 1receptor (AT1R). The inhibitor can be valsartan, olmesartan, or/andtelmisartan. The inhibitor can be an antagonist of MEK, an antagonist ofERK1,2 or/and an antagonist of STAT3. In some aspects, the disclosedinhibitor of PAX2 expression or activity is not an AT1R receptorantagonist. The term “antagonist” refers to an agent that inhibits theactivity of the target.

The antagonists of MEK and/or ERK1,2 include U0126 and PD98059. U0126 isa chemically synthesized organic compound that was initially recognizedas a cellular AP-1 antagonist, and found to be a very selective andhighly potent inhibitor of Mitogen-Activated Protein Kinase (MAPK)cascade by inhibiting its immediate upstream activators, MitogenActivated Protein Kinase Kinase 1 and 2 (also known as MEK1 and MEK2,IC50: 70 and 60 nM respectively). U0126 inhibits both active andinactive MEK1,2, unlike PD98059 which only inhibits activation ofinactive MEK. Blockade of MEK activation would prevent downstreamphosphorylation of a number of factors including p62TCF (Elk-1), anupstream inducer of c-Fos and c-Jun, components of the AP-1 complex.Inhibition of MEK/ERK pathway by U0126 also prevents all effects ofoncogenic H-Ras and K-Ras, inhibits part of the effects triggered bygrowth factors and blocks the production of inflammatory cytokines andmatrix metalloproteinases.

PD98059 has been shown to act in vivo as a highly selective inhibitor ofMEK1 activation and the MAP kinase cascade. PD98059 binds to theinactive forms of MEK1 and prevents activation by upstream activatorssuch as c-Raf. PD98059 inhibits activation of MEK1 and MEK2 with IC50values of 404 and 50 μM, respectively.

In certain embodiments, the expression of PAX2 is inhibited byadministering to the breast cancer tissue or MIN tissue in the subject ablocker of RAS signaling pathway.

In certain other embodiment, the inhibitor of PAX2 expression or PAX2activity is conjugated to an antibody, a receptor or a ligand to targetthe tumor tissue.

Enhancer of DEFB-1 expression or DEFB-1 activity.

Enhancer of DEFB-1 expression or DEFB-1 activity can be vectors thatexpress DEFB-1 protein. Since PAX2 inhibits DEFB-1 expression,inhibitors of PAX2 expression or PAX2 activity are also enhancers ofDEFB-1 expression.

Delivery Systems

There are a number of compositions and methods which can be used todeliver nucleic acids to cells, either in vitro or in vivo. Thesemethods and compositions can largely be broken down into two classes:viral based delivery systems and non-viral based delivery systems. Forexample, the nucleic acids can be delivered through a number of directdelivery systems such as, electroporation, lipofection, calciumphosphate precipitation, plasmids, viral vectors, viral nucleic acids,phage nucleic acids, phages, cosmids, or via transfer of geneticmaterial in cells or carriers such as cationic liposomes. Such methodsare well known in the art and readily adaptable for use with thecompositions and methods described herein. In certain cases, the methodswill be modified to specifically function with large DNA molecules.Further, these methods can be used to target certain diseases and cellpopulations by using the targeting characteristics of the carrier.

The inhibitors of PAX2 expression or PAX2 activity and enhancers ofDEFB1 expression or DEFB1 activity may be delivered to the target cellsusing nucleic acid based delivery systems, such as plasmids and viralvectors. As used herein, plasmid or viral vectors are agents thattransport the disclosed nucleic acids, such as PAX2 siRNA into the cellwithout degradation and include a promoter yielding expression of thegene in the cells into which it is delivered. In some embodiments thevectors are derived from either a virus or a retrovirus. Viral vectorsare, for example, Adenovirus, Adeno-associated virus, Herpes virus,Vaccinia virus, Polio virus, AIDS virus, neuronal trophic virus, Sindbisand other RNA viruses, including these viruses with the HIV backbone.Also preferred are any viral families which share the properties ofthese viruses which make them suitable for use as vectors. Retrovirusesinclude Murine Maloney Leukemia virus, MMLV, and retroviruses thatexpress the desirable properties of MMLV as a vector. Retroviral vectorsare able to carry a larger genetic payload, i.e., a transgene or markergene, than other viral vectors, and for this reason are a commonly usedvector. However, they are not as useful in non-proliferating cells.Adenovirus vectors are relatively stable and easy to work with, havehigh titers, and can be delivered in aerosol formulation, and cantransfect non-dividing cells. Pox viral vectors are large and haveseveral sites for inserting genes, they are thermostable and can bestored at room temperature. Viral vectors can have higher transaction(ability to introduce genes) abilities than chemical or physical methodsto introduce genes into cells. Typically, viral vectors contain,nonstructural early genes, structural late genes, an RNA polymerase IIItranscript, inverted terminal repeats necessary for replication andencapsidation, and promoters to control the transcription andreplication of the viral genome. When engineered as vectors, virusestypically have one or more of the early genes removed and a gene orgene/promotor cassette is inserted into the viral genome in place of theremoved viral DNA. Constructs of this type can carry up to about 8 kb offoreign genetic material. The necessary functions of the removed earlygenes are typically supplied by cell lines which have been engineered toexpress the gene products of the early genes in trans.

The nucleic acids that are delivered to cells typically containexpression controlling systems. For example, the inserted genes in viraland retroviral systems usually contain promoters, and/or enhancers tohelp control the expression of the desired gene product. A promoter isgenerally a sequence or sequences of DNA that function when in arelatively fixed location in regard to the transcription start site. Apromoter contains core elements required for basic interaction of RNApolymerase and transcription factors, and may contain upstream elementsand response elements.

Preferred promoters controlling transcription from vectors in mammalianhost cells may be obtained from various sources, for example, thegenomes of viruses such as: polyoma, Simian Virus 40 (SV40), adenovirus,retroviruses, hepatitis-B virus and most preferably cytomegalovirus, orfrom heterologous mammalian promoters, e.g. beta actin promoter.

Enhancer generally refers to a sequence of DNA that functions at nofixed distance from the transcription start site and can be either 5′ or3′ to the transcription unit. Furthermore, enhancers can be within anintron as well as within the coding sequence. They are usually between10 and 300 bp in length, and they function in cis. Enhancers f unctionto increase transcription from nearby promoters. Enhancers also oftencontain response elements that mediate the regulation of transcription.Promoters can also contain response elements that mediate the regulationof transcription. Enhancers often determine the regulation of expressionof a gene. While many enhancer sequences are now known from mammaliangenes (globin, elastase, albumin, fetoprotein and insulin), typicallyone will use an enhancer from a eukaryotic cell virus for generalexpression. Preferred examples are the SV40 enhancer on the late side ofthe replication origin (bp 100-270), the cytomegalovirus early promoterenhancer, the polyoma enhancer on the late side of the replicationorigin, and adenovirus enhancers.

The promotor and/or enhancer may be specifically activated either bylight or specific chemical events which trigger their function. Systemscan be regulated by reagents such as tetracycline and dexamethasone.There are also ways to enhance viral vector gene expression by exposureto irradiation, such as gamma irradiation, or alkylating chemotherapydrugs.

In certain embodiments the promoter and/or enhancer region can act as aconstitutive promoter and/or enhancer to maximize expression of theregion of the transcription unit to be transcribed. In certainconstructs the promoter and/or enhancer region be active in alleukaryotic cell types, even if it is only expressed in a particular typeof cell at a particular time. A preferred promoter of this type is theCMV promoter (650 bases). Other preferred promoters are SV40 promoters,cytomegalovirus (full length promoter), and retroviral vector LTR.

It has been shown that all specific regulatory elements can be clonedand used to construct expression vectors that are selectively expressedin specific cell types such as melanoma cells. The glial fibrillaryacetic protein (GFAP) promoter has been used to selectively expressgenes in cells of glial origin.

Expression vectors used in eukaryotic host cells (yeast, fungi, insect,plant, animal, human or nucleated cells) may also contain sequencesnecessary for the termination of transcription which may affect mRNAexpression. These regions are transcribed as polyadenylated segments inthe untranslated portion of the mRNA encoding tissue factor protein. The3′ untranslated regions also include transcription termination sites. Itis preferred that the transcription unit also contain a polyadenylationregion. One benefit of this region is that it increases the likelihoodthat the transcribed unit will be processed and transported like mRNA.The identification and use of polyadenylation signals in expressionconstructs is well established. It is preferred that homologouspolyadenylation signals be used in the transgene constructs. In certaintranscription units, the polyadenylation region is derived from the SV40early polyadenylation signal and consists of about 400 bases. It is alsopreferred that the transcribed units contain other standard sequencesalone or in combination with the above sequences improve expressionfrom, or stability of, the construct.

The viral vectors may include nucleic acid sequence encoding a markerproduct. This marker product is used to determine if the gene has beendelivered to the cell and once delivered is being expressed. Preferredmarker genes are the E. coli lacZ gene, which encodes ß-galactosidase,and green fluorescent protein.

In some embodiments the marker may be a selectable marker. Examples ofsuitable selectable markers for mammalian cells are dihydrofolatereductase (DHFR), thymidine kinase, neomycin, neomycin analog G418,hydromycin, and puromycin. When such selectable markers are successfullytransferred into a mammalian host cell, the transformed mammalian hostcell can survive if placed under selective pressure. There are twowidely used distinct categories of selective regimes. The first categoryis based on a cell's metabolism and the use of a mutant cell line whichlacks the ability to grow independent of a supplemented media. Twoexamples are: CHO DHFR− cells and mouse LTK− cells. These cells lack theability to grow without the addition of such nutrients as thymidine orhypoxanthine. Because these cells lack certain genes necessary for acomplete nucleotide synthesis pathway, they cannot survive unless themissing nucleotides are provided in a supplemented media. An alternativeto supplementing the media is to introduce an intact DHFR or TK geneinto cells lacking the respective genes, thus altering their growthrequirements. Individual cells which were not transformed with the DHFRor TK gene will not be capable of survival in non-supplemented media.

The second category is dominant selection which refers to a selectionscheme used in any cell type and does not require the use of a mutantcell line. These schemes typically use a drug to arrest growth of a hostcell. Those cells which have a novel gene would express a proteinconveying drug resistance and would survive the selection. Examples ofsuch dominant selection use the drugs neomycin, mycophenolic acid, orhygromycin. The three examples employ bacterial genes under eukaryoticcontrol to convey resistance to the appropriate drug G418 or neomycin(geneticin), xgpt (mycophenolic acid) or hygromycin, respectively.Others include the neomycin analog G418 and puramycin.

Non-Nucleic Acid Based Systems

The inhibitors of PAX2 expression or PAX2 activity and enhancers ofDEFB1 expression or DEFB1 activity may also be delivered to the targetcells in a variety of ways. For example, the compositions can bedelivered through electroporation, or through lipofection, or throughcalcium phosphate precipitation. The delivery mechanism chosen willdepend in part on the type of cell targeted and whether the delivery isoccurring for example in vivo or in vitro.

Thus, the compositions can comprise lipids such as liposomes, such ascationic liposomes (e.g., DOTMA, DOPE, DC-cholesterol) or anionicliposomes. Liposomes can further comprise proteins to facilitatetargeting a particular cell, if desired. Administration of a compositioncomprising a compound and a cationic liposome can be administered to theblood afferent to a target organ or inhaled into the respiratory tractto target cells of the respiratory tract. Furthermore, the compound canbe administered as a component of a microcapsule that can be targeted tospecific cell types, such as macrophages, or where the diffusion of thecompound or delivery of the compound from the microcapsule is designedfor a specific rate or dosage.

In the methods described above which include the administration anduptake of exogenous DNA into the cells of a subject (i.e., genetransduction or transfection), delivery of the compositions to cells canbe via a variety of mechanisms. As one example, delivery can be via aliposome, using commercially available liposome preparations such asLIPOFECTIN, LIPOFECTAMINE (GIBCO-BRL, Inc., Gaithersburg, Md.),SUPERFECT (Qiagen, Inc. Hilden, Germany) and TRANSFECTAM (PromegaBiotec, Inc., Madison, Wis.), as well as other liposomes developedaccording to procedures standard in the art. In addition, the disclosednucleic acid or vector can be delivered in vivo by electroporation, thetechnology for which is available from Genetronics, Inc. (San Diego,Calif.) as well as by means of a SONOPORATION machine (ImaRxPharmaceutical Corp., Tucson, Ariz.).

The materials may be in solution, suspension (for example, incorporatedinto microparticles, liposomes, or cells). These may be targeted to aparticular cell type via antibodies, receptors, or receptor ligands.Vehicles such as “stealth” and other antibody conjugated liposomes(including lipid mediated drug targeting to colonic carcinoma), receptormediated targeting of DNA through cell specific ligands, lymphocytedirected tumor targeting, and highly specific therapeutic retroviraltargeting of murine glioma cells in vivo. In general, receptors areinvolved in pathways of endocytosis, either constitutive or ligandinduced. These receptors cluster in clathrin-coated pits, enter the cellvia clathrin-coated vesicles, pass through an acidified endosome inwhich the receptors are sorted, and then either recycle to the cellsurface, become stored intracellularly, or are degraded in lysosomes.The internalization pathways serve a variety of functions, such asnutrient uptake, removal of activated proteins, clearance ofmacromolecules, opportunistic entry of viruses and toxins, dissociationand degradation of ligand, and receptor-level regulation. Many receptorsfollow more than one intracellular pathway, depending on the cell type,receptor concentration, type of ligand, ligand valency, and ligandconcentration.

Nucleic acids that are delivered to cells which are to be integratedinto the host cell genome, typically contain integration sequences.These sequences are often viral related sequences, particularly whenviral based systems are used. These viral integration systems can alsobe incorporated into nucleic acids which are to be delivered using anon-nucleic acid based system of deliver, such as a liposome, so thatthe nucleic acid contained in the delivery system can be come integratedinto the host genome.

Other general techniques for integration into the host genome include,for example, systems designed to promote homologous recombination withthe host genome. These systems typically rely on sequence flanking thenucleic acid to be expressed that has enough homology with a targetsequence within the host cell genome that recombination between thevector nucleic acid and the target nucleic acid takes place, causing thedelivered nucleic acid to be integrated into the host genome. Thesesystems and the methods necessary to promote homologous recombinationare known to those of skill in the art.

The inhibitors of PAX2 expression or PAX2 activity and enhancers ofDEFB1 expression or DEFB1 activity can be delivered to the target cellsin a variety of ways. can be administered in a pharmaceuticallyacceptable carrier and can be delivered to the subjects cells in vivoand/or ex vivo by a variety of mechanisms well known in the art (e.g.,uptake of naked DNA, liposome fusion, intramuscular injection of DNA viaa gene gun, endocytosis and the like).

If ex vivo methods are employed, cells or tissues can be removed andmaintained outside the body according to standard protocols well knownin the art. The compositions can be introduced into the cells via anygene transfer mechanism, such as, for example, calcium phosphatemediated gene delivery, electroporation, microinjection orproteoliposomes. The transduced cells can then be infused (e.g., in apharmaceutically acceptable carrier) or homotopically transplanted backinto the subject per standard methods for the cell or tissue type.Standard methods are known for transplantation or infusion of variouscells into a subject.

Composition and Kits

Another aspect of the present invention relates to compositions and kitsfor treating or preventing cancer. The composition includes an inhibitorof PAX2 expression or PAX2 activity, and/or an enhancer of DEFB-1expression or DEFB-1 activity, and a pharmaceutically acceptablecarrier.

By “pharmaceutically acceptable” is meant a material that is notbiologically or otherwise undesirable, i.e., the material may beadministered to a subject, along with the nucleic acid or vector,without causing any undesirable biological effects or interacting in adeleterious manner with any of the other components of thepharmaceutical composition in which it is contained. The carrier wouldnaturally be selected to minimize any degradation of the activeingredient and to minimize any adverse side effects in the subject, aswould be well known to one of skill in the art.

Suitable carriers and their formulations are described in Remington: TheScience and Practice of Pharmacy (19th ed.) ed. A. R. Gennaro, MackPublishing Company, Easton, Pa. 1995. Typically, an appropriate amountof a pharmaceutically-acceptable salt is used in the formulation torender the formulation isotonic. Examples of thepharmaceutically-acceptable carrier include, but are not limited to,saline, Ringer's solution and dextrose solution. The pH of the solutionis preferably from about 5 to about 8, and more preferably from about 7to about 7.5. Further carriers include sustained release preparationssuch as semipermeable matrices of solid hydrophobic polymers containingthe antibody, which matrices are in the form of shaped articles, e.g.,films, liposomes or microparticles. It will be apparent to those personsskilled in the art that certain carriers may be more preferabledepending upon, for instance, the route of administration andconcentration of composition being administered.

Pharmaceutical carriers are known to those skilled in the art. Thesemost typically would be standard carriers for administration of drugs tohumans, including solutions such as sterile water, saline, and bufferedsolutions at physiological pH. The compositions can be administeredintramuscularly or subcutaneously. Other compounds will be administeredaccording to standard procedures used by those skilled in the art.

Pharmaceutical compositions may include carriers, thickeners, diluents,buffers, preservatives, surface active agents and the like in additionto the molecule of choice. Pharmaceutical compositions may also includeone or more active ingredients such as antimicrobial agents,anti-inflammatory agents, anesthetics, and the like.

Preparations for parenteral administration include sterile aqueous ornon-aqueous solutions, suspensions, and emulsions. Examples ofnon-aqueous solvents are propylene glycol, polyethylene glycol,vegetable oils such as olive oil, and injectable organic esters such asethyl oleate. Aqueous carriers include water, alcoholic/aqueoussolutions, emulsions or suspensions, including saline and bufferedmedia. Parenteral vehicles include sodium chloride solution, Ringer'sdextrose, dextrose and sodium chloride, lactated Ringer's, or fixedoils. Intravenous vehicles include fluid and nutrient replenishers,electrolyte replenishers (such as those based on Ringer's dextrose), andthe like. Preservatives and other additives may also be present such as,for example, antimicrobials, anti-oxidants, chelating agents, and inertgases and the like.

Formulations for topical administration may include ointments, lotions,creams, gels, drops, suppositories, sprays, liquids and powders.Conventional pharmaceutical carriers, aqueous, powder or oily bases,thickeners and the like may be necessary or desirable.

Compositions for oral administration include powders or granules,suspensions or solutions in water or non-aqueous media, capsules,sachets, or tablets. Thickeners, flavorings, diluents, emulsifiers,dispersing aids or binders may be desirable.

Some of the compositions may potentially be administered as apharmaceutically acceptable acid- or base-addition salt, formed byreaction with inorganic acids such as hydrochloric acid, hydrobromicacid, perchloric acid, nitric acid, thiocyanic acid, sulfuric acid, andphosphoric acid, and organic acids such as formic acid, acetic acid,propionic acid, glycolic acid, lactic acid, pyruvic acid, oxalic acid,malonic acid, succinic acid, maleic acid, and fumaric acid, or byreaction with an inorganic base such as sodium hydroxide, ammoniumhydroxide, potassium hydroxide, and organic bases such as mono-, di-,trialkyl and aryl amines and substituted ethanolamines.

The materials may be in solution, suspension (for example, incorporatedinto microparticles, liposomes, or cells). These may be targeted to aparticular cell type via antibodies, receptors, or receptor ligands.Vehicles such as “stealth” and other antibody conjugated liposomes(including lipid mediated drug targeting to colonic carcinoma), receptormediated targeting of DNA through cell specific ligands, lymphocytedirected tumor targeting, and highly specific therapeutic retroviraltargeting of murine glioma cells in vivo. In general, receptors areinvolved in pathways of endocytosis, either constitutive or ligandinduced. These receptors cluster in clathrin-coated pits, enter the cellvia clathrin-coated vesicles, pass through an acidified endosome inwhich the receptors are sorted, and then either recycle to the cellsurface, become stored intracellularly, or are degraded in lysosomes.The internalization pathways serve a variety of functions, such asnutrient uptake, removal of activated proteins, clearance ofmacromolecules, opportunistic entry of viruses and toxins, dissociationand degradation of ligand, and receptor-level regulation. Many receptorsfollow more than one intracellular pathway, depending on the cell type,receptor concentration, type of ligand, ligand valency, and ligandconcentration.

The materials described above as well as other materials can be packagedtogether in any suitable combination as a kit useful for performing, oraiding in the performance of, the disclosed method. It is useful if thekit components in a given kit are designed and adapted for use togetherin the disclosed method. For example disclosed are kits for detecting,treating, or preventing prostate cancer, PIN, breast cancer, and MIN.The kit comprising an inhibitor of PAX2 expression or PAX2 activity,and/or an enhancer of DEFB1 expression or DEFB1 activity. In oneembodiment, the kit contains a peptide or an antibody that specificallybind PAX2 or DEFB1.

A composition disclosed herein may be administered in a number of waysdepending on whether local or systemic treatment is desired, and on thearea to be treated. For example, the compositions may be administeredorally, parenterally (e.g., intravenous, subcutaneous, intraperitoneal,or intramuscular injection), by inhalation, extracorporeally, topically(including transdermally, ophthalmically, vaginally, rectally,intranasally) or the like.

As used herein, “topical intranasal administration” means delivery ofthe compositions into the nose and nasal passages through one or both ofthe nares and can comprise delivery by a spraying mechanism or dropletmechanism, or through aerosolization of the nucleic acid or vector.Administration of the compositions by inhalant can be through the noseor mouth via delivery by a spraying or droplet mechanism. Delivery canalso be directly to any area of the respiratory system (e.g., lungs) viaintubation.

Parenteral administration of the composition, if used, is generallycharacterized by injection. Injectables can be prepared in conventionalforms, either as liquid solutions or suspensions, solid forms suitablefor solution of suspension in liquid prior to injection, or asemulsions. A more recently revised approach for parenteraladministration involves use of a slow release or sustained releasesystem such that a constant dosage is maintained.

The exact amount of the compositions required will vary from subject tosubject, depending on the species, age, weight and general condition ofthe subject, the severity of the allergic disorder being treated, theparticular nucleic acid or vector used, its mode of administration andthe like. An appropriate amount can be determined by one of ordinaryskill in the art using only routine experimentation given the teachingsherein. Thus, effective dosages and schedules for administering thecompositions may be determined empirically, and making suchdeterminations is within the skill in the art. The dosage ranges for theadministration of the compositions are those large enough to produce thedesired effect in which the symptoms disorders are affected. The dosageshould not be so large as to cause adverse side effects, such asunwanted cross-reactions, anaphylactic reactions, and the like.Generally, the dosage will vary with the age, condition, sex and extentof the disease in the patient, route of administration, or whether otherdrugs are included in the regimen, and can be determined by one of skillin the art. The dosage can be adjusted by the individual physician inthe event of any counter indications. Dosage can vary, and can beadministered in one or more dose administrations daily, for one orseveral days. Guidance can be found in the literature for appropriatedosages for given classes of pharmaceutical products.

For example, a typical daily dosage of the disclosed composition usedalone might range from about 1 μg/kg to up to 100 mg/kg of body weightor more per day, depending on the factors mentioned above. In certainembodiments, the treatment method is tailored based on the PAX2-to-DEFB1expression ratio (P/D ratio) and estrogen-receptor(ER)/progesterone-receptor (PR) status of the diseased tissue. Table 2shows the treatment options based on the P/D ratio and ER/PR status.There is a positive correlation between PAX2 status and ER status innormal breast tissue, MIN and low grade breast carcinoma. PAX2 alsoregulates ERBB2 expression and subsequently Her2/neu expression via theoestogen receptor. Conversely, there is an inverse relationship betweenPAX2 expression and high grade (or invasive) breast carcinoma. Thereforemonitoring PAX2 expression levels can be used to predict drug responseor resistance, as well as identify patients who may be candidates forDEFB1 or anti-PAX2 therapy. The term “anti-PAX2 therapy” refers tomethods for inhibiting PAX2 expression or PAX2 activity. The term “DEFB1therapy” refers to methods for increasing DEFB1 expression. The term“DEFB1 therapy” does not include methods for inhibiting PAX2 expressionor PAX2 activity, although such methods also result in increase of DEFB1expression.

As shown in Table 2, anti-PAX2 therapy and/or DFB1 therapy may be usedin conjunction with one or more other treatments for breast cancer, suchas anti-hormone treatment (e.g., Tamoxifen), anti-ERBB2 treatment (e.g.,Herceptin), anti-Her2 treatment (e.g., Trastuzumab), andanti-AIB-1/SRC-3 treatment.

TABLE 2 Using PAX2-to-DEFB1 Ratio to Treat Breast Conditions Change inPAX2/ Anti- Tissue DEFB ER/PR DEFB1 PAX2 Type 1 Ratio* Status TherapyTherapy Adjuvant Therapy MIN

ER⁺/PR⁺ No Yes No Low Grade ER⁻/PR⁺ Yes Yes Anti-ERBB2 Cancer (eg.Herceptin) Anti-Her2 (eg. Trastuzumab) Anti-AIB-1/SRC-3 Low Grade

ER⁺/PR⁻ Yes Yes Anti-ERBB2 Cancer (eg. Herceptin) Anti-Her2 (eg.Trastuzumab) Anti-AIB-1/SRC-3 High Grade

ER⁺/PR⁺ Yes No Anti-hormone Cancer (eg. Tamoxifen) Anti-ERBB2 (eg.Herceptin) Anti-Her2 (eg. Trastuzumab) Anti-AIB-1/SRC-3 High Grade

ER⁺/PR⁻ Yes No Anti-hormone Cancer (eg. Tamoxifen) Anti-ERBB2 (eg.Herceptin) Anti-Her2 (eg. Trastuzumab) Anti-AIB-1/SRC-3 High Grade

ER⁻/PR⁺ Yes No Anti-ERBB2 Cancer (eg. Herceptin) Anti-Her2 (eg.Trastuzumab) High Grade

ER⁻/PR⁻ Yes No Anti-ERBB2 Cancer (eg. Herceptin) Anti-Her2 (eg.Trastuzumab) *Compared to the PAX2/DEFB1 ratio in normal breastepithelium

PAX2-to-DEFB1 Expression Ratio

As used hereinafter, the term “PAX2-to-DEFB1 expression ratio” refers tothe ratio between the amount of functional PAX2 protein or its variantand the amount of functional DEFB1 protein or its variant in a givencell or tissue. Levels of PAX2 and DEFB1 expression in a cell or tissuecan be measured any method known in the art. In certain embodiments, thelevels of PAX2 and DEFB1 expression in breast tissue are determined bydetermining the levels of PAX2 and DEFB1 in a cell or cells obtaineddirectly from the breast tissue.

The “PAX2-to-DEFB1 expression ratio” can be determined directly atprotein level or indirectly at the RNA level. The protein levels may bemeasured with protein arrays, immunoassays and enzyme assays. The RNAlevels may be measured, for example, with DNA arrays, RT-PCR andNorthern Blotting. In certain embodiments, the PAX2-to-DEFB1 expressionratio is determined by determining the expression level of PAX2 generelative to the expression level of a control gene, determining theexpression level of DEFB1 gene relative to the expression level of thesame control gene, and calculating the PAX2-to-DEFB1 expression ratiobased on the expression levels of PAX2 and DEFB1. In one embodiment, thecontrol gene is the glyceraldehyde 3-phosphate dehydrogenase (GAPDH)gene.

Immunoassays

Immunoassays, in their most simple and direct sense, are binding assaysinvolving binding between antibodies and antigen. Many types and formatsof immunoassays are known and all are suitable for detecting thedisclosed biomarkers. Examples of immunoassays are enzyme linkedimmunosorbent assays (ELISAs), radioimmunoassays (RIA), radioimmuneprecipitation assays (RIPA), immunobead capture assays, Westernblotting, dot blotting, gel-shift assays, Flow cytometry, proteinarrays, multiplexed bead arrays, magnetic capture, in vivo imaging,fluorescence resonance energy transfer (FRET), and fluorescencerecovery/localization after photobleaching (FRAP/FLAP).

In general, immunoassays involve contacting a sample suspected ofcontaining a molecule of interest (such as the disclosed biomarkers)with an antibody to the molecule of interest or contacting an antibodyto a molecule of interest (such as antibodies to the disclosedbiomarkers) with a molecule that can be bound by the antibody, as thecase may be, under conditions effective to allow the formation ofimmunocomplexes. In many forms of immunoassay, the sample-antibodycomposition, such as a tissue section, ELISA plate, dot blot or Westernblot, can then be washed to remove any non-specifically bound antibodyspecies, allowing only those antibodies specifically bound within theprimary immune complexes to be detected.

Radioimmune Precipitation Assay (RIPA) is a sensitive assay usingradiolabeled antigens to detect specific antibodies in serum. Theantigens are allowed to react with the serum and then precipitated usinga special reagent such as, for example, protein A sepharose beads. Thebound radiolabeled immunoprecipitate is then commonly analyzed by gelelectrophoresis. Radioimmunoprecipitation assay (RIPA) is often used asa confirmatory test for diagnosing the presence of HIV antibodies. RIPAis also referred to in the art as Fan Assay, Precipitin Assay,Radioimmune Precipitin Assay; Radioimmunoprecipitation Analysis;Radioimmunoprecipitation Analysis, and RadioimmunoprecipitationAnalysis.

Also contemplated are immunoassays wherein the protein or antibodyspecific for the protein is bound to a solid support (e.g., tube, well,bead, or cell) to capture the antibody or protein of interest,respectively, from a sample, combined with a method of detecting theprotein or antibody specific for the protein on the support. Examples ofsuch immunoassays include Radioimmunoassay (RIA), Enzyme-LinkedImmunosorbent Assay (ELISA), Flow cytometry, protein array, multiplexedbead assay, and magnetic capture.

Protein arrays are solid-phase ligand binding assay systems usingimmobilized proteins on surfaces which include glass, membranes,microtiter wells, mass spectrometer plates, and beads or otherparticles. The assays are highly parallel (multiplexed) and oftenminiaturized (microarrays, protein chips). Their advantages includebeing rapid and automatable, capable of high sensitivity, economical onreagents, and giving an abundance of data for a single experiment.Bioinformatics support is important; the data handling demandssophisticated software and data comparison analysis. However, thesoftware can be adapted from that used for DNA arrays, as can much ofthe hardware and detection systems.

Capture arrays form the basis of diagnostic chips and arrays forexpression profiling. They employ high affinity capture reagents, suchas conventional antibodies, single domains, engineered scaffolds,peptides or nucleic acid aptamers, to bind and detect specific targetligands in high throughput manner. Antibody arrays are availablecommercially. In addition to the conventional antibodies, Fab and scFvfragments, single V-domains from camelids or engineered humanequivalents (Domantis, Waltham, Mass.) may also be useful in arrays.

Nonprotein capture molecules, notably the single-stranded nucleic acidaptamers which bind protein ligands with high specificity and affinity,are also used in arrays (SomaLogic, Boulder, Colo.). Aptamers areselected from libraries of oligonucleotides by the Selex™ procedure andtheir interaction with protein can be enhanced by covalent attachment,through incorporation of brominated deoxyuridine and UV-activatedcrosslinking (photoaptamers). Photocrosslinking to ligand reduces thecrossreactivity of aptamers due to the specific steric requirements.Aptamers have the advantages of ease of production by automatedoligonucleotide synthesis and the stability and robustness of DNA; onphotoaptamer arrays, universal fluorescent protein stains can be used todetect binding.

An alternative to an array of capture molecules is one made through‘molecular imprinting’ technology, in which peptides (e.g., from theC-terminal regions of proteins) are used as templates to generatestructurally complementary, sequence-specific cavities in apolymerizable matrix; the cavities can then specifically capture(denatured) proteins that have the appropriate primary amino acidsequence (ProteinPrint™, Aspira Biosystems, Burlingame, Calif.).

Another methodology which can be used diagnostically and in expressionprofiling is the ProteinChip® array (Ciphergen, Fremont, Calif.), inwhich solid phase chromatographic surfaces bind proteins with similarcharacteristics of charge or hydrophobicity from mixtures such as plasmaor tumor extracts, and SELDI-TOF mass spectrometry is used to detectionthe retained proteins.

Other useful methodology includes large-scale functional chipsconstructed by immobilizing large numbers of purified proteins on achip, and multiplexed bead assays.

Antibodies

The term “antibodies” is used herein in a broad sense and includes bothpolyclonal and monoclonal antibodies. In addition to intactimmunoglobulin molecules, also included in the term “antibodies” arefragments or polymers of those immunoglobulin molecules, and human orhumanized versions of immunoglobulin molecules or fragments thereof, aslong as they are chosen for their ability to interact with, for example,PAX2 or DEFB1, such that PAX2 is inhibited from interacting with DEFB1.Antibodies that bind the disclosed regions of PAX2 or DEFB1 involved inthe interaction between PAX2 and DEFB1 are also disclosed. Theantibodies can be tested for their desired activity using the in vitroassays described herein, or by analogous methods, after which their invivo therapeutic and/or prophylactic activities are tested according toknown clinical testing methods.

The monoclonal antibodies herein specifically include “chimeric”antibodies in which a portion of the heavy and/or light chain isidentical with or homologous to corresponding sequences in antibodiesderived from a particular species or belonging to a particular antibodyclass or subclass, while the remainder of the chain(s) is identical withor homologous to corresponding sequences in antibodies derived fromanother species or belonging to another antibody class or subclass, aswell as fragments of such antibodies, as long as they exhibit thedesired antagonistic activity (See, U.S. Pat. No. 4,816,567 and Morrisonet al., Proc. Natl. Acad. Sci. USA, 81:6851-6855 (1984)).

As used herein, the term “antibody” or “antibodies” can also refer to ahuman antibody and/or a humanized antibody. Many non-human antibodies(e.g., those derived from mice, rats, or rabbits) are naturallyantigenic in humans, and thus can give rise to undesirable immuneresponses when administered to humans. Therefore, the use of human orhumanized antibodies in the methods serves to lessen the chance that anantibody administered to a human will evoke an undesirable immuneresponse. Methods for humanizing non-human antibodies are well known inthe art.

DNA Arrays

A DNA or oligonucleotide microarray consists of an arrayed series of aplurality of microscopic spots of oligonucleotides, called features,each containing a small amount (typically in the range of picomoles) ofa specific oligonucleotide sequence. The specific oligonucleotidesequence can be a short section of a gene or other oligonucleotideelement that are used as probes to hybridize a cDNA or cRNA sample underhigh-stringency conditions. Probe-target hybridization is usuallydetected and quantified by fluorescence-based detection offluorophore-labeled targets to determine relative abundance of nucleicacid sequences in the target.

The probes are typically attached to a solid surface by a covalent bondto a chemical matrix (via epoxy-silane, amino-silane, lysine,polyacrylamide or others). The solid surface can be glass or a siliconchip or microscopic beads. Oligonucleotide arrays are different fromother types of microarray only in that they either measure nucleotidesor use oligonucleotide as part of its detection system.

To detect gene expression in target tissue or cells using anoligonucleotide array, nucleic acid of interest is purified from thetarget tissue or cells. The nucleotide can be all RNA for expressionprofiling, DNA for comparative hybridization, or DNA/RNA bound to aparticular protein which is immunoprecipitated (ChIP-on-chip) forepigenetic or regulation studies.

In one embodiment, total RNA is isolated (total as it is nuclear andcytoplasmic) by guanidinium thiocyanate-phenol-chloroform extraction(e.g. Trizol). The purified RNA may be analyzed for quality (e.g., bycapillary electrophoresis) and quantity (e.g., by using a nanodropspectrometer. The total RNA is RNA is reverse transcribed into DNA witheither polyT primers or random primers. The DNA products may beoptionally amplified by PCR. A label is added to the amplificationproduct either in the RT step or in an additional step afteramplification if present. The label can be a fluorescent label orradioactive labels. The labeled DNA products are then hybridized to themicroarray. The microarray is then washed and scanned. The expressionlevel of the gene of interest is determined based on the hybridizationresult using method well known in the art.

Pharmacogenomics

In another embodiment, the PAX2 and/or DEFB1 expression profiles areused for determine pharmacogenomics of breast cancer. Pharmacogenomicsrefers to the relationship between an individual's genotype and thatindividual's response to a foreign compound or drug. Differences inmetabolism of therapeutics can lead to severe toxicity or therapeuticfailure by altering the relation between dose and blood concentration ofthe pharmacologically active drug. Thus, a physician or clinician mayconsider applying knowledge obtained in relevant pharmacogenomicsstudies in determining whether to administer an anti-cancer drug, aswell as tailoring the dosage and/or therapeutic regimen of treatmentwith the anti-cancer drug.

Pharmacogenomics deals with clinically significant hereditary variationsin the response to drugs due to altered drug disposition and abnormalaction in affected persons. In general, two types of pharmacogeneticconditions can be differentiated. Genetic conditions transmitted as asingle factor altering the way drugs act on the body (altered drugaction) or genetic conditions transmitted as single factors altering theway the body acts on drugs (altered drug metabolism). Thesepharmacogenetic conditions can occur either as rare genetic defects oras naturally-occurring polymorphisms. For example, glucose-6-phosphatedehydrogenase deficiency (G6PD) is a common inherited enzymopathy inwhich the main clinical complication is hemolysis after ingestion ofoxidant drugs (anti-malarials, sulfonamides, analgesics, nitrofurans)and consumption of fava beans.

One pharmacogenomics approach to identifying genes that predict drugresponse, known as “a genome-wide association,” relies primarily on ahigh-resolution map of the human genome consisting of already knowngene-related sites (e.g., a “bi-allelic” gene marker map which consistsof 60,000-100,000 polymorphic or variable sites on the human genome,each of which has two variants). Such a high-resolution genetic map canbe compared to a map of the genome of each of a statisticallysubstantial number of subjects taking part in a Phase II/III drug trialto identify genes associated with a particular observed drug response orside effect. Alternatively, such a high resolution map can be generatedfrom a combination of some ten-million known single nucleotidepolymorphisms (SNPs) in the human genome. As used herein, an “SNP” is acommon alteration that occurs in a single nucleotide base in a stretchof DNA. For example, an SNP may occur once per every 1,000 bases of DNA.An SNP may be involved in a disease process. However, the vast majorityof SNPs may not be disease associated. Given a genetic map based on theoccurrence of such SNPs, individuals can be grouped into geneticcategories depending on a particular pattern of SNPs in their individualgenome. In such a manner, treatment regimens can be tailored to groupsof genetically similar individuals, taking into account traits that maybe common among such genetically similar individuals. Thus, mapping ofthe PAX2 and/or DEFB1 to SNP maps of breast patients may allow easieridentification of these genes according to the genetic methods describedherein.

Alternatively, a method termed the “candidate gene approach,” can beutilized to identify genes that predict drug response. According to thismethod, if a gene that encodes a drug target is known, all commonvariants of that gene can be fairly easily identified in the populationand it can be determined if having one version of the gene versusanother is associated with a particular drug response.

As an illustrative embodiment, the activity of drug metabolizing enzymesis a major determinant of both the intensity and duration of drugaction. The discovery of genetic polymorphisms of drug metabolizingenzymes (e.g., N-acetyltransferase 2 (NAT 2) and cytochrome P450 enzymesCYP2D6 and CYPZC19) has provided an explanation as to why some subjectsdo not obtain the expected drug effects or show exaggerated drugresponse and serious toxicity after taking the standard and safe dose ofa drug. These polymorphisms are expressed in two phenotypes in thepopulation, the extensive metabolizer and poor metabolizer. Theprevalence of poor metabolizer phenotypes is different among differentpopulations. For example, the gene coding for CYP2D6 is highlypolymorphic and several mutations have been identified in poormetabolizers, which all lead to the absence of functional CYP2D6. Poormetabolizers of CYP2D6 and CYP2C19 quite frequently experienceexaggerated drug response and side effects when they receive standarddoses. If a metabolite is the active therapeutic moiety, poormetabolizers show no therapeutic response, as demonstrated for theanalgesic effect of codeine mediated by its CYP2D6-formed metabolitemorphine. The other extreme are the so called ultra-rapid metabolizerswho do not respond to standard doses. Recently, the molecular basis ofultra-rapid metabolism has been identified to be due to CYP2D6 geneamplification.

Alternatively, a method termed the “gene expression profiling” can beutilized to identify genes that predict drug response. For example, thegene expression of an animal dosed with a drug can give an indicationwhether gene pathways related to toxicity have been turned on.

Information generated from more than one of the above pharmacogenomicsapproaches can be used to determine appropriate dosage and treatmentregimens for prophylactic or therapeutic treatment an individual. Thisknowledge, when applied to dosing or drug selection, can avoid adversereactions or therapeutic failure and thus enhance therapeutic orprophylactic efficiency when treating a subject with a breast condition.

In one embodiment, the PAX2 and/or DEFB1 expression profiles, as well asthe ER/PR status, in a subject are used to determine the appropriatetreatment regimens for an individual with a breast condition.

In another embodiment, the PAX2 expression level (typically determine inreference to a control gene as actin gene or GAPDH gene) is used inpatients with triple negative breast cancer (i.e., oestrogen receptor(ER) negative, progesterone receptor (PR) negative, human epidermalgrowth factor receptor 2 (HER2) negative) to measure of theeffectiveness of cancer therapy, to determine treatment course, or tomonitor cancer recurrence.

The present invention is further illustrated by the following exampleswhich should not be construed as limiting. The contents of allreferences, patents and published patent applications cited throughoutthis application, as well as the Figures and Tables are incorporatedherein by reference.

Example 1: Human Beta Defensin-1 is Cytotoxic to Late-Stage ProstateCancer and Plays a Role in Prostate Cancer Tumor Immunity

In this example, DEFB1 was cloned into an inducible expression system toexamine what effect it had on normal prostate epithelial cells, as wellas androgen receptor positive (AR+) and androgen receptor negative (AR−)prostate cancer cell lines. Induction of DEFB1 expression resulted in adecrease in cellular growth in AR− cells DU145 and PC3, but had noeffect on the growth of the AR+prostate cancer cells LNCaP. DEFB1 alsocaused rapid induction of caspase-mediated apoptosis. Data presentedhere are the first to provide evidence of its role in innate tumorimmunity and indicate that its loss contributes to tumor progression inprostate cancer.

Materials and Methods

Cell Lines: The cell lines DU145 were cultured in DMEM medium, PC3 weregrown in F12 medium, and LNCaP were grown in RPMI medium (LifeTechnologies, Inc., Grand Island, N.Y.). Growth media for all threelines was supplemented with 10% (v/v) fetal bovine serum (LifeTechnologies). The hPrEC cells were cultured in prostate epitheliumbasal media (Cambrex Bio Science, Inc., Walkersville, Md.). All celllines were maintained at 37° C. and 5% CO2.

Tissue Samples and Laser Capture Microdissection: Prostate tissuesobtained from consented patients that underwent radical prostatectomywere acquired through the Hollings Cancer Center tumor bank inaccordance with an Institutional Review Board-approved protocol. Thisincluded guidelines for the processing, sectioning, histologicalcharacterization, RNA purification and PCR amplification of samples.Following pathologic examination of frozen tissue sections, lasercapture microdissection (LCM) was performed to ensure that the tissuesamples assayed consisted of pure populations of benign prostate cells.For each tissue section analyzed, LCM was performed at three differentregions containing benign tissue and the cells collected were thenpooled.

Prostate tissues were obtained from patients who provided informedconsent prior to undergoing radical prostatectomy. Samples were acquiredthrough the Hollings Cancer Center tumor bank in accordance with anInstitutional Review Board-approved protocol. This included guidelinesfor the processing, sectioning, histological characterization, RNApurification and PCR amplification of samples. Prostate specimensreceived from the surgeons and pathologists were immediately frozen inOCT compound. Each OCT block was cut to produce serial sections whichwere stained and examined. Areas containing benign cells, prostaticintraepithelial neoplasia (PIN), and cancer were identified and used toguide our selection of regions from unstained slides using the ArcturusPixCell II System (Sunnyvale, Calif.). Caps containing captured materialwere exposed to 20 μl of lysate from the Arcturus Pico Pure RNAIsolation Kit and processed immediately. RNA quantity and quality wasevaluated using sets of primers that produce 5′ amplicons. The setsinclude those for the ribosomal protein L32 (the 3′ amplicon and the 5′amplicon are 298 bases apart), for the glucose phosphate isomerase (391bases apart), and for the glucose phosphate isomerase (842 bases apart).Ratios of 0.95 to 0.80 were routinely obtained for these primer setsusing samples from a variety of prepared tissues. Additional tumor andnormal samples were grossly dissected by pathologists, snap frozen inliquid nitrogen and evaluated for hBD-1 and cMYC expression.

Cloning of DEFBJ Gene: DEFB1 cDNA was generated from RNA by reversetranscription-PCR. The PCR primers were designed to contain ClaI andKpnI restriction sites. DEFB1 PCR products were restriction digestedwith ClaI and KpnI and ligated into a TA cloning vector. The TA/DEFB1vector was then transfected into E. coli by heat shock and individualclones were selected and expanded. Plasmids were isolated by CellCulture DNA Midiprep (Qiagen, Valencia, Calif.) and sequence integrityverified by automated sequencing. The DEFB1 gene fragment was thenligated into the pTRE2 digested with ClaI and KpnI, which served as anintermediate vector for orientation purposes. Then the pTRE2/DEFB1construct was digested with ApaI and KpnI to excise the DEFB1 insert,which was ligated into pIND vector of the Ecdysone Inducible ExpressionSystem (Invitrogen, Carlsbad, Calif.) also double digested with ApaI andKpnI. The construct was again transfected into E. coli and individualclones were selected and expanded. Plasmids were isolated and sequenceintegrity of pIND/DEFB1 was again verified by automated sequencing.

Transfection: Cells (1×10⁶) were seeded onto 100-mm Petri dishes andgrown overnight. Then the cells were co-transfected using Lipofectamine2000 (Invitrogen, Carlsbad, Calif.) with 1 μg of pVgRXR plasmid, whichexpresses the heterodimeric ecdysone receptor, and 1 μg of thepIND/DEFB1 vector construct or empty pIND control vector in Opti-MEMmedia (Life Technologies, Inc., Grand Island, N.Y.).

RNA Isolation and Quantitative RT-PCR: In order to verify DEFB1 proteinexpression in the cells transfected with DEFB1 construct, RNA wascollected after a 24 hour induction period with Ponasterone A (Pon A).Briefly, total RNA was isolated using the SV Total RNA Isolation System(Promega, Madison, Wis.) from approximately 1×10⁶ cells harvested bytrypsinizing. Here, cells were lysed and total RNA was isolated bycentrifugation through spin columns. For cells collected by LCM, totalRNA was isolated using the PicoPure RNA Isolation Kit (ArcturusBiosciences, Mt. View, Calif.) following the manufacturer's protocol.Total RNA (0.5 μg per reaction) from both sources was reversetranscribed into cDNA utilizing random primers (Promega). AMV ReverseTranscriptase II enzyme (500 units per reaction; Promega) was used forfirst strand synthesis and Tfl DNA Polymerase for second strandsynthesis (500 units per reaction; Promega) as per the manufacturer'sprotocol. In each case, 50 pg of cDNA was used per ensuing PCR reaction.Two-step QRT-PCR was performed on cDNA generated using the MultiScribeReverse Transcripatase from the TaqMan Reverse Transcription System andthe SYBR® Green PCR Master Mix (Applied Biosystems).

The primer pair for DEFB1 was generated from the published DEFB1sequence (GenBank Accession No. U50930). The primer sequences are:

Sense (5′-3′) β-actin SEQ ID NO: 51 5′-CCTGGCACCCAGCACAAT-3′ DEFB1SEQ ID NO: 53 5′-GTTGCCTGCCAGTCGCCATGAGAACTTCCTAC-3′ Antisense (5′-3′)β-actin SEQ ID NO: 52 5′-GCCGATCCACACGGAGTACT-3′ DEFB1 SEQ ID NO: 545′-TGGCCTTCCCTCTGTAACAGGTGCCTTGAATT-3′

Forty cycles of PCR were performed under standard conditions using anannealing temperature of 56° C. In addition, β-actin (Table 2) wasamplified as a housekeeping gene to normalize the initial content oftotal cDNA. DEFB1 expression was calculated as the relative expressionratio between DEFB1 and β-actin and was compared in cells lines inducedand uninduced for DEFB1 expression, as well as LCM benign prostatictissue. As a negative control, QRT-PCR reactions without cDNA templatewere also performed. All reactions were run three times in triplicate.

MIT Cell Viability Assay: To examine the effects of DEFB1 on cellgrowth, metabolic 3-[4,5-dimethylthiazol-2yl]-2,5 diphenyl tetrazoliumbromide (MTT) assays were performed. PC3, DU145 and LNCaP cellsco-transfected with pVgRXR plasmid and pIND/DEFB1 construct or emptypIND vector were seeded onto a 96-well plate at 1-5×10³ cells per well.Twenty-four hours after seeding, fresh growth medium was addedcontaining 10 μM Ponasterone A daily to induce DEFB1 expression for 24-,48- and 72 hours after which the MTT assay was performed according tothe manufacturer's instructions (Promega). Reactions were performedthree times in triplicate.

Flow Cytometry: PC3 and DU145 cells co-transfected with the DEFB1expression system were grown in 60-mm dishes and induced for 12, 24, and48 hours with 10 μM Ponasterone A. Following each incubation period, themedium was collected from the plates (to retain any detached cells) andcombined with PBS used to wash the plates. The remaining attached cellswere harvested by trypsinization and combined with the detached cellsand PBS. The cells were then pelleted at 4° C. (500×g) for 5 mM, washedtwice in PBS, and resuspended in 100 ul of 1× Annexin binding buffer(0.1 M Hepes/NaOH at pH 7.4, 1.4 M NaCl, 25 mM CaCl₂) containing 5 μl ofAnnexin V-FITC and 5 μl of PI. The cells were incubated at RT for 15 mMin the dark, then diluted with 400 μl of 1× Annexin binding buffer andanalyzed by FACscan (Becton Dickinson, San Jose, Calif.). All reactionswere performed three times.

Microscopic Analysis: Cell morphology was analyzed by phase contrastmicroscopy. DU145, PC3 and LNCaP cells containing no vector, emptyplasmid or DEFB1 plasmid were seeded onto 6 well culture plates (BDFalcon, USA). The following day plasmid-containing cells were inducedfor a period of 48 h with media containing 10 μM Ponasterone A, whilecontrol cells received fresh media. The cells were then viewed under aninverted Zeiss IM 35 microscope (Carl Zeiss, Germany). Phase contrastpictures of a field of cells were obtained using the SPOT Insight Mosaic4.2 camera (Diagnostic Instruments, USA). Cells were examined by phasecontrast microscopy under 32× magnification and digital images werestored as uncompressed TIFF files and exported into Photoshop CSsoftware (Adobe Systems, San Jose, Calif.) for image processing and hardcopy presentation.

Caspase Detection: Detection of caspase activity in the prostate cancercell lines was performed using APO LOGIX™ Carboxyfluorescin Caspasedetection kit (Cell Technology, Mountain View, Calif.). Active caspaseswere detected through the use of a FAM-VAD-FMK inhibitor thatirreversibly binds to active caspases. Briefly, DU145 and PC3 cells(1.5-3×10⁵) containing the DEFB1 expression system were plated in 35 mmglass bottom microwell dishes (Matek, Ashland, Mass.) and treated for 24hours with media only or with media containing PonA as previouslydescribed. Next, 10 μl of a 30× working dilution of carboxyfluoresceinlabeled peptide fluoromethyl ketone (FAM-VAD-FMK) was added to 300 μl ofmedia and added to each 35 mm dish. Cells were then incubated for 1 hourat 37° C. under 5% CO2. Then, the medium was aspirated and the cellswere washed twice with 2 ml of a 1× Working dilution Wash Buffer. Cellswere viewed under differential interference contrast (DIC) or underlaser excitation at 488 nm. The fluorescent signal was analyzed using aconfocal microscope (Zeiss LSM 5 Pascal) and a 63×DIC oil lens with aVario 2 RGB Laser Scanning Module.

Statistical Analysis: Statistical differences were evaluated using theStudent's t-test for unpaired values. P values were determined by atwo-sided calculation, and a P value of less than 0.05 was consideredstatistically significant.

Results

DEFB1 Expression in Prostate Tissue and Cell Lines: DEFB1 expressionlevels were measured by QRT-PCR in benign and malignant prostatictissue, hPrEC prostate epithelial cells and DU145, PC3 and LNCaPprostate cancer cells. DEFB1 expression was detected in all of thebenign clinical samples. The average amount of DEFB1 relative expressionwas 0.0073. In addition, DEFB1 relative expression in hPrEC cells was0.0089. There was no statistical difference in DEFB1 expression detectedin the benign prostatic tissue samples and hPrEC (FIG. 1A). Analysis ofthe relative DEFB1 expression levels in the prostate cancer cell linesrevealed significantly lower levels in DU145, PC3 and LNCaP. As afurther point of reference, relative DEFB1 expression was measured inthe adjacent malignant section of prostatic tissue from patient #1215.There were no significant differences in the level of DEFB1 expressionobserved in the three prostate cancer lines compared to malignantprostatic tissue from patient #1215 (FIG. 1B). In addition, expressionlevels in all four samples were close to the no template negativecontrols which confirmed little to no endogenous DEFB1 expression (datanot shown). QRT-PCR was also performed on the prostate cancer cell linestransfected with the DEFB1 expression system. Following a 24 hourinduction period, relative expression levels were 0.01360 in DU145,0.01503 in PC3 and 0.138 in LNCaP. Amplification products were verifiedby gel electrophoresis.

QRT-PCR was performed on LCM tissues regions containing benign, PIN andcancer. DEFB1 relative expression was 0.0146 in the benign regioncompared to 0.0009 in the malignant region (FIG. 1C). This represents a94% decrease which again demonstrates a significant down-regulation ofexpression. Furthermore, analysis of PIN revealed that DEFB1 expressionlevel was 0.044 which was a 70% decrease. Comparing expression inpatient #1457 to the average expression level found in benign regions ofsix other patients (FIG. 1A) revealed a ratio of 1.997 representingalmost twice as much expression (FIG. 1D). However, the expression ratiowas 0.0595 in PIN and was 0.125 in malignant tissue compared to averageexpression levels in benign tissue.

DEFB1 Causes Cell Membrane Permeability and Ruffling: Induction of DEFB1in the prostate cancer cell lines resulted in a significant reduction incell number in DU145 and PC3, but had no effect on cell proliferation inLNCaP (FIG. 2). As a negative control, cell proliferation was monitoredin all three lines containing empty plasmid. There were no observablechanges in cell morphology in DU145, PC3 or LNCaP cells following theaddition of PonA. In addition, DEFB1 induction resulted in morphologicalchanges in both DU145 and PC3. Here cells appeared more rounded andexhibited membrane ruffling indicative of cell death. Apoptotic bodieswere also present in both lines.

Expression of DEFB1 Results in Decreased Cell Viability: The MTT assayshowed a reduction in cell viability by DEFB1 in PC3 and DU145 cells,but no significant effect on LNCaP cells (FIG. 3). After 24 hours,relative cell viability was 72% in DU145 and 56% in PC3. Analysis 48hours after induction revealed 49% cell viability in DU145 and 37% cellviability in PC3. After 72 hours of DEFB1 expression resulted in 44% and29% relative cell viability in DU145 and PC3 cells, respectively.

DEFB1 Causes Rapid Caspase-mediated Apoptosis in Late-stage ProstateCancer Cells: In order to determine whether the effects of DEFB1 on PC3and DU145 were cytostatic or cytotoxic, FACS analysis was performed.Under normal growth conditions, more than 90% of PC3 and DU145 cultureswere viable and non-apoptotic (lower left quadrant) and did not stainwith annexin V or PI. After inducing DEFB1 expression in PC3 cells, thenumber of apoptotic cells (lower and upper right quadrants) totaled 10%at 12 hours, 20% at 24 hours, and 44% at 48 hours (FIG. 4B). For DU145cells, the number of apoptotic cells totaled 12% after 12 hours, 34% at24 hours, and 59% after 48 hours of induction (FIG. 4A). There was noincrease in apoptosis observed in cells containing empty plasmidfollowing induction with PonA (data not shown).

Caspase activity was determined by confocal laser microscopic analysis(FIGS. 5A-5L). DU145 and PC3 cell were induced for DEFB1 expression andactivity was monitored based on the binding of green fluorescingFAM-VAD-FMK to caspases in cells actively undergoing apoptosis. Analysisof cells under DIC showed the presence of viable control DU145 (FIG.5A), PC3 (FIG. 5E) and LNCaP (FIG. 1) cells at 0 hours. Excitation bythe confocal laser at 488 nm produced no detectable green staining whichindicates no caspase activity in DU145 (FIG. 5B), PC3 (FIG. 5F) or LNCaP(FIG. 5J). Following induction for 24 hours, DU145 (FIG. 5C), PC3 (FIG.5G) and LNCaP (FIG. 5K) cells were again visible under DIC. Confocalanalysis under fluorescence revealed green staining in DU145 (FIG. 5D)and PC3 (FIG. 5H) cell indicating caspase activity. However, there wasno green staining in LNCaP (FIG. 5L), indicating no induction ofapoptosis by DEFB1.

In conclusion, this study provides the functional role of DEFB1 inprostate cancer. Furthermore, these findings show that DEFB1 is part ofan innate immune system involved in tumor immunity. Data presented heredemonstrate that DEFB1 expressed at physiological levels is cytotoxic toAR− hormone refractory prostate cancer cells, but not to AR+ hormonesensitive prostate cancer cell nor to normal prostate epithelial cells.Given that DEFB1 is constitutively expressed in normal prostate cellswithout cytotoxicity, it may be that late-stage AR− prostate cancercells possess distinct phenotypic characteristics that render themsensitive to DEFB1 cytotoxicity. Thus, DEFB1 is a viable therapeuticagent for the treatment of late-stage prostate cancer, and potentiallyother cancers as well.

Example 2: siRNA Mediated Knockdown of PAX2 Expression Results inProstate Cancer Cell Death Independent of P53 Status

This example examines the effects of inhibiting PAX2 expression by RNAinterference in prostate cancer cells which differ in p53 gene status.The results demonstrate that the inhibition of PAX2 results in celldeath irrespective of p53 status, indicating that there are additionaltumor suppressor genes or cell death pathways inhibited by PAX2 inprostate cancer.

Materials and Methods

siRNA Silencing of PAX2: In order to achieve efficient gene silencing, apool of four complementary short interfering ribonucleotides (siRNAs)targeting human PAX2 mRNA (Accession No. NM_003989.1), were synthesized(Dharmacon Research, Lafayette, Colo., USA). A second pool of foursiRNAs were used as an internal control to test for the specificity ofPAX2 siRNAs. Two of the sequences synthesized target the GL2 luciferasemRNA (Accession No. X65324), and two were non-sequence-specific (Table3). For annealing of siRNAs, 35 M of single strands were incubated inannealing buffer (100 mM potassium acetate, 30 mM HEPES-KOH at pH 7.4, 2mM magnesium acetate) for 1 min at 90° C. followed by 1 h incubation at37° C.

TABLE 3 PAX2 siRNA Sequences. A pool of four siRNA was utilized toinhibit PAX2 protein expression. Sense (5′-3′) Sequence A 5′- GAAGUCAAGUSEQ ID NO: 7 CGAGUCUAUUU-3′ Sequence B 5′-GAGGAAACGU SEQ ID NO: 8GAUGAAGAUUU-3′ Sequence C 5′-GGACAAGAUU SEQ ID NO: 9 GCUGAAUACUU-3′Sequence D 5′-CAUCAGAGCAC SEQ ID NO: 10 AUCAAAUCUU-3′ Antisense (5′-3′)Sequence A 5′-AUAGACUCGACUU SEQ ID NO: 3 GACUUCUU-3′ Sequence B5′-AUCUUCAUCACG SEQ ID NO: 4 UUUCCUCUU-3′ Sequence C 5′-GUAUUCAGCAAUSEQ ID NO: 5 CUUGUCCUU-3′ Sequence D 5′-GAUUUGAUGUGC SEQ ID NO: 6UCUGAUGUU-3′

Western Analysis: Briefly, cells were harvested by trypsinization andwashed twice with PBS. Lysis buffer was prepared according to themanufacturer's instructions (Sigma), and was then added to the cells.Following a 15 minute incubation period at 4° C. on an orbital shaker,cell lysate were then collected and centrifuged for 10 minutes at12000×g to pellet cellular debris. The protein-containing supernatantwere then collected and quantitated. Next, 25 μg protein extract wasloaded onto an 8-16% gradient SDS-PAGE (Novex). Followingelectrophoresis, proteins were transferred to PVDF membranes, and thenblocked with 5% nonfat dry milk in TTBS (0.05% Tween 20 and 100 mMTris-Cl) for 1 hour. Blots were then probed with rabbit anti-PAX2primary antibody (Zymed, San Francisco, Calif.) at a 1:2000 dilution.After washing, the membranes were incubated with anti-rabbit antibodyconjugated to horseradish peroxidase (HRP) (dilution 1:5000; Sigma), andsignal detection was visualized using chemilluminescence reagents(Pierce) on an Alpha Innotech Fluorchem 8900. As a control, blots werestripped and reprobed with mouse anti-β-actin primary antibody (1:5000;Sigma-Aldrich) and HRP-conjugated anti-mouse secondary antibody (1:5000;Sigma-Aldrich) and signal detection was again visualized.

Phase Contrast Microscopy: The effect of PAX2 knock-down on cell growthwas analyzed by phase contrast microscopy as described in Example 1.

MIT Cytotoxicity Assay: DU145, PC3 and LNCaP cells (1×10⁵) weretransfected with 0.5 μg of the PAX2 siRNA pool or control siRNA poolusing Codebreaker transfection reagent according to the manufacturer'sprotocol (Promega). Next, cell suspensions were diluted and seeded ontoa 96-well plate at 1-5×10³ cells per well and allowed to grow for 2-, 4-or 6 days. After culture, cell viability was determined by measuring theconversion of 3-[4,5-dimethylthiazol-2yl]-2,5 diphenyl tetrazoliumbromide, MTT (Promega), to a colored formazan product. Absorbance wasread at 540 nm on a scanning multiwell spectrophotometer.

Pan-Caspase Detection: Detection of caspase activity in the prostatecancer cell lines was performed s described in Example 1.

Quantitative Real-time RT-PCR: Quantitative real-time RT-PCR wasperformed as described in Example 1 in order to verify gene expressionafter PAX2 siRNA treatment in PC3, DU145 and LNCaP cell lines. Theprimer pairs for GAPDH (control gene), BAX, BID and BAD are:

Sense (5′-3′) GAPDH 5′-CCACCCATGGCAAA SEQ ID NO: 55 TTCCATGGCA-3′ BAD5′-CTCAGGCCTATGCA SEQ ID NO: 57 AAAAGAGGA-3′ BID 5′-AACCTACGCACCTSEQ ID NO: 59 ACGTGAGGAG-3′ BAX 5′-GACACCTGAGCTG SEQ ID NO: 61ACCTTGG-3′ Antisense (5′-3′) GAPDH 5′-TCTAGACGGCAGGT SEQ ID NO: 56CAGGTCAACC-3′ BAD 5′-GCCCTCCCTCCAA SEQ ID NO: 58 AGGAGAC-3′ BID5′-CGTTCAGTCCATC SEQ ID NO: 60 CCATTTCTG-3′ BAX 5′-GAGGAAGTCCAGTSEQ ID NO: 62 GTCCAGC-3′

Reactions were performed in MicroAmp Optical 96-well Reaction Plate (PEBiosystems). Forty cycles of PCR were performed under standardconditions using an annealing temperature of 60° C. Quantification wasdetermined by the cycle number where exponential amplification began(threshold value) and averaged from the values obtained from thetriplicate repeats. There was an inverse relationship between messagelevel and threshold value. In addition, GAPDH was used as a housekeepinggene to normalize the initial content of total cDNA. Gene expression wascalculated as the relative expression ratio between the pro-apoptoticgenes and GAPDH. All reactions were carried out in triplicate.

Results

siRNA Inhibition of PAX2 Protein: In order to confirm that the siRNAeffective targeted the PAX2 mRNA, Western Analysis was performed tomonitor PAX2 protein expression levels over a six day treatment period.Cells were given a single round of transfection with the pool of PAX2siRNA. The results confirmed specific targeting of PAX2 mRNA by showingknock-down of PAX2 protein by day four in DU145 (FIG. 6A) and by day sixin PC3 (FIG. 6B).

Knock-down of PAX2 inhibit Prostate Cancer Cell Growth: Cells wereanalyzed following a six day treatment period with media only, negativecontrol non-specific siRNA or PAX2 siRNA (FIG. 7). DU145 (FIG. 7A), PC3(FIG. 7D) and LNCaP (FIG. 7G) cells all reached at least 90% confluencyin the culture dishes containing media only. Treatment of DU145 (FIG.7B), PC3 (FIG. 7E) and LNCaP (FIG. 7H) with negative controlnon-specific siRNA had no effect on cell growth, and cells again reachedconfluency after six days. However, treatment with PAX2 siRNA resultedin a significant decrease in cell number. DU145 cells were approximately15% confluent (FIG. 7C) and PC3 cells were only 10% confluent (FIG. 7F).LNCaP cell were 5% confluent following siRNA treatment.

Cytotoxicity Assays: Cell viability was measured after two-, four-, andsix-day exposure times, and is expressed as a ratio of the 570-630 nmabsorbance of treated cells divided by that of the untreated controlcells (FIG. 8). Relative cell viability following 2 days of treatmentwas 77% in LNCaP, 82% in DU145 and 78% in PC3. After four days, relativecell viability was 46% in LNCaP, 53% in DU145 and 63% in PC3. After sixdays of treatment, relative cell viability decreased to 31% in LNCaP,37% in PC3, and was 53% in DU145. As negative controls, cell viabilitywas measured in after a six day treatment period with negative controlnon-specific siRNA or transfection reagent alone. For both conditions,there was no statistically significant change in cell viability comparedto normal growth media.

Pan-Caspase Detection: Caspase activity was detected by confocal lasermicroscopic analysis. DU145, PC3 and LNCaP cells were treated with PAX2siRNA and activity was monitored based on the binding of FAM-labeledpeptide to caspases in cells actively undergoing apoptosis which willfluoresce green. Analysis of cells with media only under DIC shows thepresence of viable DU145 (FIG. 9A), PC3 (FIG. 9E) and LNCaP (FIG. 9I)cells at 0 hours (FIG. 9). Excitation by the confocal laser at 488 nmproduced no detectable green staining which indicates no caspaseactivity in untreated DU145 (FIG. 9B), PC3 (FIG. 9F) or LNCaP (FIG. 9J).Following four days of treatment with PAX2 siRNA, DU145 (FIG. 9C), PC3(FIG. 9G) and LNCaP (FIG. 9K) cells were again visible under DIC. Underfluorescence, the treated DU145 (FIG. 9D), PC3 (FIG. 9H) and LNCaP (FIG.9L) cells presented green staining indicating caspase activity.

Effect of PAX2 Inhibition on Pro-apoptotic Factors: DU145, PC3 and LNCaPcells were treated with siRNA against PAX2 for six days and expressionof pro-apoptotic genes dependent and independent of p53 transcriptionregulation were measured to monitor cell death pathways. For BAX, therewas a 1.81-fold increase in LNCaP, a 2.73-fold increase in DU145, and a1.87-fold increase in PC3 (FIG. 10A). Expression levels of BID increasedby 1.38-fold in LNCaP and 1.77-fold in DU145 (FIG. 10B). However, BIDexpression levels decreased by 1.44-fold in PC3 following treatment(FIG. 10C). Analysis of BAD revealed a 2.0-fold increase in expressionin LNCaP, a 1.38-fold increase in DU145, and a 1.58-fold increase inPC3.

These results demonstrate dependency of prostate cancer cell survival onPAX2 expression. Following p53 activation as a result of PAX2 knock-downin the p53-expressing cell line LNCaP, the p53-mutated line DU145, andthe p53-null line PC3, caspase activity was detected in all three lines,indicating of the initiation of programmed cell death. BAX expressionwas upregulated in all three cell lines independent of p53 status. Theexpression of pro-apoptotic factor BAD was also increased in all threelines following PAX2 inhibition. Following treatment with PAX2 siRNA,BID expression was increased in LNCaP and DU145, but actually decreasedin PC3. These results indicate that cell death observed in prostatecancer is influenced by but is not dependent on p53 expression. Theinitiation of apoptosis in prostate cancer cells through different celldeath pathways irrespective of p53 status indicates that PAX2 inhibitsother tumor suppressors.

Example 3: Inhibition of PAX2 Oncogene Results in DEFB1-Mediated Deathof Prostate Cancer Cells

The identification of tumor-specific molecules that serve as targets forthe development of new cancer drugs is considered to be a major goal incancer research. Example 1 demonstrated that there is a high frequencyof DEFB1 expression loss in prostate cancer, and that induction of DEFB1expression results in rapid apoptosis in androgen receptornegative-stage prostate cancer. These data show that DEFB1 plays a rolein prostate tumor suppression. In addition, given that it is a naturallyoccurring component of the immune system of normal prostate epithelium,DEFB1 is expected to be a viable therapeutic agent with little to noside effects. Example 2 demonstrated that inhibition of PAX2 expressionresults in prostate cancer cell death independent of p53. These dataindicate that there is an addition pro-apoptotic factor or tumorsuppressor that is inhibited by PAX2. In addition, the data show thatthe oncogenic factor PAX2, which is over-expressed in prostate cancer,is a transcriptional repressor of DEFB1. The purpose of this study is todetermine if loss of DEFB1 expression is due to aberrant expression ofthe PAX2 oncogene, and whether inhibiting PAX2 results in expression ofDEFB1 and DEFB1-mediated cell death (FIG. 11).

Materials and Methods

RNA Isolation and Quantitative RT-PCR: RNA isolation and quantitativeRT-PCR of DEFB1 were performed as described in Example 1.

Generation of the DEFB1 Reporter Construct: The pGL3 luciferase reporterplasmid was used to monitor DEFB1 reporter activity. Here, a region 160bases upstream of the DEFB1 transcription initiation site and includedthe DEFB1 TATA box. The region also included the CCTTG (SEQ ID NO: 1)sequence which is necessary for PAX2 binding. The PCR primers weredesigned to contain KpnI and Nhe1 restriction sites. The DEFB1 promoterPCR products were restriction digested Kpn I and NheI and ligated into asimilarly restriction digested pGL3 plasmid (FIG. 12). The constructswere transfected into E. coli and individual clones were selected andexpanded. Plasmids were isolated and sequence integrity of theDEFB1/pGL3 construct was verified by automated sequencing.

Luciferase Reporter Assay: Here, 1 μg of the DEFB1 reporter construct orthe control pGL3 plasmid was transfected into 1×10⁶ DU145 cells. Next,0.5×103 cells were seeded onto each well of a 96-well plate and allowedto grow overnight. Then fresh medium was added containing PAX2 siRNA ormedia only and the cells were incubated for 48 hours. Luciferase wasdetected by the BrightGlo kit according to the manufacturer's protocol(Promega) and the plates were read on a Veritas automated 96-wellluminometer. Promoter activity was expressed as relative luminescence.

Analysis of Membrane Permeability: Acridine orange (AO)/ethidium bromide(EtBr) dual staining was performed to identify changes in cell membraneintegrity, as well as apoptotic cells by staining the condensedchromatin. AO stains viable cells as well as early apoptotic cells,whereas EtBr stains late stage apoptotic cells that have lost membranepermeability. Briefly, cells were seeded into 2 chamber culture slides(BD Falcon, USA). Cells transfected with empty pIND plasmid/pvgRXR orpIND DEFB1/pvgRXR were induced for 24 or 48 h with media containing 10μM Ponasterone A. Control cells were provided fresh media at 24 and 48h. In order to determine the effect of PAX2 inhibition on membraneintegrity, separate culture slides containing DU145, PC3 and LNCaP weretreated with PAX2 siRNA and incubated for 4 days. Following this, cellswere washed once with PBS and stained with 2 ml of a mixture (1:1) of AO(Sigma, USA) and EtBr (Promega, USA) (5 ug/ml) solution for 5 minFollowing staining, the cells were again washed with PBS. Fluorescencewas viewed by a Zeiss LSM 5 Pascal Vario 2 Laser Scanning ConfocalMicroscope (Carl Zeiss Jena, Germany). The excitation color wheelcontain BS505-530 (green) and LP560 (red) filter blocks which allowedfor the separation of emitted green light from AO into the green channeland red light from EtBr into the red channel. The laser power output andgain control settings within each individual experiment were identicalbetween control and DEFB1 induced cells. The excitation was provided bya Kr/Ar mixed gas laser at wavelengths of 543 nm for AO and 488 nm forEtBr. Slides were analyzed under 40× magnification and digital imageswere stored as uncompressed TIFF files and exported into Photoshop CSsoftware (Adobe Systems, San Jose, Calif.) for image processing and hardcopy presentation.

ChIP Analysis of PAX2: Chromatin immunoprecipitation (ChIP) allows theidentification of binding sites for DNA-binding proteins based upon invivo occupancy of a promoter by a transcription factor and enrichment oftranscription factor bound chromatin by immunoprecipitation. Amodification of the protocol described by the Farnham laboratory wasused; also on line at http://mcardle.oncology.wisc.edu/farnham/). TheDU145 and PC3 cell lines over-expresses the PAX2 protein, but does notexpress DEFB1. Cells were incubated with PBS containing 1.0%formaldehyde for 10 minutes to crosslink proteins to DNA. Samples werethen sonicated to yield DNA with an average length of 600 bp. Sonicatedchromatin precleared with Protein A Dynabeads was incubated withPAX2-specific antibody or “no antibody” control [isotype-matched controlantibodies]. Washed immunoprecipitates were then collected. Afterreversal of the crosslinks, DNA was analyzed by PCR usingpromoter-specific primers to determine whether DEFB1 is represented inthe PAX2-immunoprecipitated samples. Primers were designed to amplifythe 160 bp region immediately upstream of the DEFB1 mRNA start sitewhich contained the DEFB1 TATA box and the functional CCTTG (SEQ IDNO: 1) PAX2 recognition site. For these studies, positive controlsincluded PCR of an aliquot of the input chromatin (prior toimmunoprecipitation, but crosslinks reversed). All steps were performedin the presence of protease inhibitors.

Results

siRNA Inhibition of PAX2 Increases DEFB1 Expression: QRT-PCR analysis ofDEFB1 expression before siRNA treatment revealed relative expressionlevels of 0.00097 in DU145, 0.00001 in PC3, and 0.00004 LNCaP (FIG. 13).Following siRNA knock-down of PAX2, relative expression was 0.03294(338-fold increase) in DU145, 0.00020 (22.2-fold increase) in PC3 and0.00019 (4.92-fold increase) in LNCaP. As a negative control, the humanprostate epithelial cell line (hPrEC) which is PAX2 null, revealedexpression levels at 0.00687 before treatment and 0.00661 followingsiRNA treatment confirming no statistical change in DEFB1 expression.

siRNA Inhibition of PAX2 Increases DEFB1 Promoter Activity: FIG. 14shows that inhibition of PAX2 results in increased DEFB1 promoteractivity. PC3 promoter/pGL3 and DU145 promoter/pGL3 construct weregenerated and were transfected into PC3 and DU145 cells, respectively.Promoter activity was compared before and after PAX2 inhibition by siRNAtreatment. DEFB1 promoter activity increased 2.65-fold in DU145 and 3.78fold in PC3 following treatment.

DEFB1 Causes Cell Membrane Permeability: Membrane integrity wasmonitored by confocal analysis. As shown in FIG. 15, intact cells staingreen due to AO which is membrane permeable. In addition, cells withcompromised plasma membranes would stain red by EtBr which is membraneimpermeable. Here, uninduced DU145 (FIG. 5A) and PC3 (FIG. 15D) cellsstained positively with AO and emitted green color, but did not stainwith EtBr. However, DEFB1 induction in both DU145 (FIG. 15B) and PC3(FIG. 15E) resulted in the accumulation of EtBr in the cytoplasm at 24hours indicated by the red staining. By 48 hours, DU145 (FIG. 15C) andPC3 (FIG. 15F) possessed condensed nuclei and appeared yellow, which wasdue to the presence of both green and red staining resulting from theaccumulation of AO and EtBr, respectively.

Inhibition of PAX2 Results in Membrane Permeability: Cells were treatedwith PAX2 siRNA for 4 days and membrane integrity was monitored again byconfocal analysis. As shown in FIG. 16, both DU145 and PC3 possessedcondensed nuclei and appeared yellow. However, LNCaP cells' cytoplasmand nuclei remained green following siRNA treatment. Also red stainingat the cell periphery indicates the maintenance of cell membraneintegrity. These findings indicate that the inhibition of PAX2 resultsin specifically DEFB1-mediated cell death in DU1145 and PC3, but notLNCaP cells. Death observed in LNCaP is due to the transactivation ofthe existing wild-type p53 in LNCap following PAX2 inhibition.

PAX2 Binds to the DEFB1 Promoter: ChIP analysis was performed on DU145and PC3 cells to determine if the PAX2 transcriptional repressor isbound to the DEFB1 promoter (FIG. 17). Lane 1 contains a 100 bpmolecular weight marker. Lane 2 is a positive control representing 160bp region of the DEFB1 promoter amplified from DU145 beforecross-linking and immunoprecipitation. Lane 3 is a negative controlrepresenting PCR performed without DNA. Lanes 4 and 5 are negativecontrols representing PCR from immunoprecipitations performed with IgGfrom cross-linked DU145 and PC3, respectively. PCR amplification of 25pg of DNA (lane 6 and 8) and 50 pg of DNA (lane 7 and 9)immunoprecitipated with anti-PAX2 antibody after crosslinking show 160bp promoter fragment in DU145 and PC3, respectively.

FIG. 18 shows predicted structure of the PrdPD and PrdHD with DNA. Thecoordinates of the structures of the PrdPD bound to DNA (Xu et al.,1995) and the PrdHD bound to DNA (Wilson et al., 1995) were used toconstruct a model of the two domains as they bound to a PHO site. Theindividual binding sites are abutted next to each other with a specificorientation as indicated. The RED domain is oriented based on the PrdPDcrystal structure.

FIG. 19 shows comparison of consensus sequences of different paireddomains. At the top of the Figure is drawn a schematic representation ofprotein±DNA contacts described in the crystallographic analysis of thePrd-paired-domain±DNA complex. Empty boxes indicate a-helices, shadedboxes indicates b-sheets and a thick line indicate a b-turn. Contactingamino acids are shown by single-letter code. Only direct amino acid±basecontacts are shown. Empty circles indicate major groove contacts whilered arrows indicate minor groove contacts. This scheme is aligned to allknown consensus sequences for paired-domain proteins (top strands onlyare shown). Vertical lines between consensus sequences indicateconserved base-pairs. Numbering of the positions is shown at the bottomof the Figure.

These results demonstrate that the oncogenic factor PAX2 suppressesDEFB1 expression. The suppression occurs at the transcriptional level.Furthermore, computational analysis of the DEFB1 promoter revealed thepresence of a CCTTG (SEQ ID NO: 1) DNA binding site for the PAX2transcriptional repressor next to the DEFB1 TATA box (FIG. 1). One ofthe hallmarks of defensin cytotoxicity is the disruption of membraneintegrity. These results show that ectopic expression of DEFB1 inprostate cancer cells results in a loss of membrane potential due tocompromised cell membranes. The same phenomenon is observed afterinhibiting PAX2 protein expression. Therefore, suppression of PAX2expression or function, results in the re-establishment of DEFB1expression and subsequently DEFB1-mediated cell death. Also, the presentresults establish the utility of DEFB1 as a directed therapy forprostate cancer treatment, and potentially other cancer treatments,through innate immunity.

Example 4: Effect of Defb1 Expression in Implanted Tumor Cells

The anti-tumoral ability of DEFB1 is evaluated by injecting tumor cellsthat overexpress DEFB1 into nude mice. DEFB1 is cloned into pBI-EGFPvector, which has a bidirectional tetracycline responsible promoter.Tet-Off Cell lines are generated by transfecting pTet-Off into DU145,PC3 and LNCaP cells and selecting with G418. The pBI-EGFP-DEFB1 plasmidis co-transfected with pTK-Hyg into the Tet-off cell lines and selectedwith hygromycin. Only single-cell suspensions with a viability of >90%are used. Each animal receives approximately 500,000 cells administeredsubcutaneously into the right flank of female nude mice. There are twogroups, a control group injected with vector only clones and a groupinjected with the DEFB1 over-expressing clones. 35 mice are in eachgroup as determined by a statistician. Animals are weighed twice weekly,tumor growth monitored by calipers and tumor volumes determined usingthe following formula: volume=0.5×(width)2×length. All animals aresacrificed by CO2 overdose when tumor size reaches 2 mm3 or 6 monthsfollowing implantation; tumors are excised, weighed and stored inneutral buffered formalin for pathological examination. Differences intumor growth between the groups are descriptively characterized throughsummary statistics and graphical displays. Statistical significance isevaluated with either the t-test or non-parametric equivalent.

Example 5: Effect of PAX2 siRNA on Implanted Tumor Cells

Hairpin PAX2 siRNA template oligonucleotides utilized in the in vitrostudies are utilized to examine the effect of the up-regulation of DEFB1expression in vivo. The sense and antisense strand (see Table 3) areannealed and cloned into pSilencer 2.1 U6 hygro siRNA expression vector(Ambion) under the control of the human U6 RNA pol III promoter. Thecloned plasmid is sequenced, verified and transfected into PC3, Du145,and LNCap cell lines. Scrambled shRNA is cloned and used as a negativecontrol in this study. Hygromycin resistant colonies are selected, cellsare introduced into the mice subcutaneously and tumor growth ismonitored as described above.

Example 6: Effect of Small Molecule Inhibitors of PAX2 Binding onImplanted Tumor Cells

The DNA recognition sequence for PAX2 binding resides in the DEFB1promoter between nucleotides −75 and −71 (+1 refers to thetranscriptional start site). Short oligonucleotides complementary to thePAX2 DNA-binding domain are provided. Examples of such oligonucleotidesinclude the 20-mer and 40-mer oligonucleotides containing the CCTTG (SEQID NO: 1) recognition sequence provided below. These lengths wererandomly selected, and other lengths are expected to be effective asblockers of binding. As a negative control, oligonicleotides with ascrambled sequence (CTCTG)-(SEQ ID NO: 22) were designed to verifyspecificity. The oligonucleotides are transfected into the prostatecancer cells and the HPrEC cells with lipofectamine reagent orCodebreaker transfection reagent (Promega, Inc). In order to confirmDNA-protein interactions, double stranded oligonucleotides will belabeled with [³²1] dCTP and electrophoretic mobility shift assays areperformed. In addition, DEFB1 expression is monitored by QRT-PCR andWestern analysis following treatment with oligonucleotides. Finally,cell death is detected by MTT assay and flow cytometry as previouslydescribed.

Recognition Sequence #1: (SEQ ID NO: 18) CTCCCTTCAGTTCCGTCGACRecognition Sequence #2:  (SEQ ID NO: 19) CTCCCTTCACCTTGGTCGACScramble Sequence #1: (SEQ ID NO: 23) CTCCCTTCACTCTGGTCGACRecognition Sequence #3: (SEQ ID NO: 20)ACTGTGGCACCTCCCTTCAGTTCCGTCGACGAGGTTGTGC Recognition Sequence #4:(SEQ ID NO: 21) ACTGTGGCACCTCCCTTCACCTTGGTCGACGAGGTTGTGCScramble Sequence #2: (SEQ ID NO: 24)ACTGTGGCACCTCCCTTCACTCTGGTCGACGAGGTTGTGC

Further examples of oligonucleotides of the invention include:

Recognition Sequence #1: (SEQ ID NO: 25) 5′-AGAAGTTCACCCTTGACTGT-3′Recognition Sequence #2: (SEQ ID NO: 26) 5′-AGAAGTTCACGTTCCACTGT-3′Scramble Sequence #1: (SEQ ID NO: 27) 5′-AGAAGTTCACGCTCTACTGT-3′Recognition Sequence #3: (SEQ ID NO: 28) 5′-TTAGCGATTAGAAGTTCACCCTTGACTGTGGCACCTCCC-3′ Recognition Sequence #4: (SEQ ID NO: 29)5′-GTTAGCGATTAGAAGTTCACGTTC CACTGTGGCACCTCCC-3′ Scramble Sequence #2: (SEQ ID NO: 30) 5′-GTTAGCGATTAGAAGTTCACGCT CTACTGTGGCACCTCCC-3′

This set of alternative inhibitory oligonucleotides represents therecognition sequence for the PAX2 binding domain and homeobox. Theseinclude actual sequences from the DEFB1 promoter.

The PAX2 gene is required for the growth and survival of various cancercells including prostate. In addition, the inhibition of PAX2 expressionresults in cell death mediated by the innate immunity component DEFB1.Suppression of DEFB1 expression and activity is accomplished by bindingof the PAX2 protein to a CCTTG (SEQ ID NO: 1) recognition site in theDEFB1 promoter. Therefore, this pathway provides a viable therapeutictarget for the treatment of prostate cancer. In this method, thesequences bind to the PAX2 DNA binding site and block PAX2 binding tothe DEFB1 promoter thus allowing DEFB1 expression and activity. Theoligonucleotide sequences and experiment described above are examples ofand demonstrate a model for the design of additional PAX2 inhibitordrugs.

Given that the CCTTG (SEQ ID NO: 1) sequence exists in interleukin-3,interleukin-4, the insulin receptor and others, PAX2 regulates theirexpression and activity as well. Therefore the PAX2 inhibitors disclosedherein have utility in a number of other diseases including thosedirected related to inflammation including prostatitis and benignprostatic hypertrophy (BPH).

Example 7: Loss of DEFB1 Expression Results in Increased Tumorigenesis

Generation of Loss of Function Mice: The Cre/loxP system has been usefulin elucidating the molecular mechanisms underlying prostatecarcinogenesis. Here a DEFB1 Cre conditional KO is used for inducibledisruption within the prostate. The DEFB1 Cre conditional KO involvesthe generation of a targeting vector containing loxP sites flankingDEFB1 coding exons, targeted ES cells with this vector and thegeneration of germline chimeric mice from these targeted ES cells.Heterozygotes are mated to prostate-specific Cre transgenics andheterozygous intercross is used to generate prostate-specific DEFB1 KOmice. Four genotoxic chemical compounds have been found to induceprostate carcinomas in rodents: N-methyl-N-nitrosourea (MNU),N-nitrosobis 2-oxopropyl amine (BOP), 3,2X-dimethyl-4-amino-biphenyl(MAB) and 2-amino-1-methyl-6-phenylimidazow 4,5-bxpyridine (PhIP).DEFB1-transgenic mice are treated with these carcinogenic compounds viaintra-gastric administration or i.v. injection for prostate adenoma andadenocarcinoma induction studies. Prostate samples are studied fordifferences in tumor growth and changes gene expression thoughhistological, immunohistological, mRNA and protein analyses.

Generation of GOF mice: For PAX2 inducible GOF mice, PAX2 GOF(bi-transgenic) and wild-type (mono-transgenic) littermates areadministered doxycycline (Dox) from 5 weeks of age to induceprostate-specific PAX2 expression. Briefly, PROBASIN-rtTAmono-transgenic mice (prostate cell-specific expression of tet-dependentrtTA inducer) are crossed to our PAX2 transgenic responder lines. Forinduction, bi-transgenic mice are fed Dox via the drinking water (500mg/L freshly prepared twice a week). Initial experiments verify lowbackground levels, good inducibility and cell-type specific expressionof PAX2 and the EGFP reporter using transgenic founder line inbi-transgenic mice. Regarding experimental group sizes, 5-7 age- andsex-matched individuals in each group (wild-type and GOF) allow forstatistical significance. For all animals in this study, prostatetissues are collected initially at weekly intervals for analysis andcomparison, to determine carcinogenic time parameters. PCR Genotyping,RT-PCR and qPCR: PROBASIN-rtTA transgenic mice are genotyped using thefollowing PCR primers and conditions: PROBASINS (forward)5′-ACTGCCCATTGCCCAAACAC-3′ (SEQ ID NO: 31);

RTTA3 (reverse) 5′-AAAATCTTGCCAGCTTTCCCC-3′ (SEQ ID NO: 32); 95° C.denaturation for 5 min, followed by 30 cycles of 95° C. for 30 sec, 57°C. for 30 sec, 72° C. for 30 sec, followed by a 5 min extension at 72°C., yielding a 600 bp product. PAX2 inducible transgenic mice aregenotyped using the following PCR primers and conditions: PAX2For5′-GTCGGTTACGGAGCGGACCGGAG-3′ (SEQ ID NO: 33);Rev5′IRES 5′-TAACATATAGACAAACGCACACCG-3′ (SEQ ID NO: 34); 95° C.denaturation for 5 min, followed by 34 cycles of 95° C. for 30 sec, 63°C. for 30 sec, 72° C. for 30 sec, followed by a 5 min extension at 72°C., yielding a 460 bp product.

Immortomouse hemizygotes are be genotyped using the following PCRprimers and conditions: Immol1, 5′-GCGCTTGTGTC GCCATTGTATTC-3′ (SEQ IDNO: 35); Immol2, 5′-GTCACACCACAGAAGTAAGGTTCC-3′ (SEQ ID NO: 36); 94° C.30 sec, 58° C. 1 min, 72° C. 1 min 30 sec, 30 cycles to yield a ˜1 kbtransgene band. For genotyping PAX2 knockout mice, the following PCRprimers and conditions are used: PAX2 For 5′-GTCGGTTACGGAGCGGACCGGAG-3′(SEQ ID NO: 37); PAX2Rev 5′-CACAGAGCATTGGCGATCTCGATGC-3′ (SEQ ID NO:38); 94° C. 1 min, 65° C. 1 min, 72° C. 30 sec, 36 cycles to yield a 280bp band.

DEFB1 Peptide Animal Studies: Six-week-old male athymic (nude) micepurchased from Charles River Laboratories are injected sub-cutaneouslyover the scapula with 10⁶ viable PC3 cells. One week after injection,the animals are randomly allocated to one of three groups—group I:control; group II: intraperitoneal injections of DEFB1, 100 μg/day, 5days a week, for weeks 2-14; group III: intraperitoneal injections ofDEFB1, 100 mg/day, 5 days a week, for weeks 8-14. Animals are maintainedin sterile housing, four animals to a cage, and observed on a dailybasis. At 10-day intervals, the tumors are measured by using calipers,and the volumes of the tumors are calculated by using V=(L×W2)/2.

Example 8: Targeting PAX2 Expression for the Chemoprevention ofIntraepithelial Neoplasia and Cancer

Cancer chemoprevention is defined as the prevention of cancer ortreatment at the pre-cancer state or even earlier. The long period ofprogression to invasive cancer is a major scientific opportunity butalso an economic obstacle to showing the clinical benefit of candidatechemopreventive drugs. Therefore, an important component ofchemopreventive agent development research in recent years has been toidentify earlier (than cancer) end points or biomarkers that accuratelypredict an agent's clinical benefit or cancer incidence-reducing effect.In many cancers, IEN is an early end point such as in prostate cancer.Given that the PAX2/DEFB1 pathway is deregulated during IEN and perhapsat even an earlier histopathological state makes it a powerfulpredictive biomarker and an excellent target for chemoprevention ofcancer. Shown are a number of compounds that suppress PAX2 and increasesDEFB1 expression that may have utility as chemoprevention agents forprostate cancer.

As shown in Table 1, the PAX2 gene is expressed in a number of cancers.In addition, several cancers have been shown to have aberrant PAX2expression (FIG. 20). Angiotensin II (AngII) is a major regulator ofblood pressure and cardiovascular homeostasis and is recognized as apotent mitogen. AngII mediates its biological effects through binding totwo subtypes of receptors, Angiotensin Type I receptor (AT1R) andAngiotensin Type II receptor (AT2R) which belong to the super-family ofG-protein-coupled receptors but have different tissue distribution andintracellular signaling pathways. In addition to its effects on bloodpressure, AngII has been shown to play a role in various pathologicalsituations involving tissue remodeling, such as wound healing, cardiachypertrophy and development. In fact, recent studies have revealed localexpression of several components of the Renin-Angiotensin System (RAS)in various cancer cells and tissues including the prostate. Upregulationof AT1R provides a considerable advantage to cancer cells that havelearn to evade apoptosis and growth regulatory elements. To date anumber of cancers have been shown to aberrantly express PAX2.Chemoprevention via target PAX2 expression may have a significant impacton cancer related deaths.

Materials and Methods

Cell Culture: The cell lines DU145, LnCap and PC3 were cultured asdescribed in Eaxmple 1. The hPrEC cells were cultured in prostateepithelium basal media (Cambrex Bio Science, Inc., Walkersville, Md.)and maintained at 37° C. and 5% CO2.

Reagents and Treatments: Cells were treated with 5 or 10 uM of AngII, 5uM of the ATR1 antagonist Los, 5 uM of the ATR2 antagonist PD123319, 25uM of the MEK inhibitor U0126, 20 uM of the MEK/ERK inhibitor PD98059 or250 μM of the AMP kinase inducer AICAR.

Western Analysis: Western blot was performed as described in Eaxmple 2.Blots were then probed with primary antibody (anti-PAX2, -phospho-PAX2,-JNK, -phospho-JNK, -ERK1/2, or -phospho-ERK1/2) (Zymed, San Francisco,Calif.) at 1:1000-2000 dilutions. After washing, the membranes wereincubated with anti-rabbit antibody conjugated to horseradish peroxidase(HRP) (dilution 1:5000; Sigma), and signal detection was visualizedusing chemilluminescence reagents (Pierce) on an Alpha InnotechFluorchem 8900. As a control, blots were stripped and re-probed withmouse anti-β-actin primary antibody (1:5000; Sigma-Aldrich) andHRP-conjugated anti-mouse secondary antibody (1:5000; Sigma-Aldrich),and signal detection was again visualized.

QRT-PCR Analysis: Quantitative real-time RT-PCR was performed asdescribed in Example 1 to verify changes in gene expression followingPAX2 knockdown in PC3 and DU145 prostate cancer cell lines and the hPrECnormal prostate epithelial cells. Forty cycles of PCR were performedunder standard conditions using an annealing temperature of 60° C.Quantification was determined by the cycle number where exponentialamplification began (threshold value) and averaged from the valuesobtained from the triplicate repeats. There was an inverse relationshipbetween message level and threshold value. In addition, GAPDH was usedas a housekeeping gene to normalize the initial content of total cDNA.Relative expression was calculated as the ratio between each genes andGAPDH. All reactions were carried out in triplicate.

Thymidine Incorporation: Proliferation of cells was determined by [³H]thymidine ribotide ([3H] TdR) incorporation into DNA. 0.5×106 cells/wellof suspension DU145 cells were plated in their appropriate media. Cellswere incubated for 72 h with or without the presence of AngII at theindicated concentrations. Cells were exposed to 37 kBq/ml [methyl-3H]thymidine in the same medium for 6 h. The adherent cells were fixed by5% trichloroacetic acid and lysed in SDS/NaOH lysis buffer overnight.Radioactivity was measured by Beckman LS3801 liquid scintillationcounter (Canada). Suspension cell culture was harvested by cellharvester (Packard instrument Co., Meriden, Conn.), and radioactivitywas measured by 1450 microbeta liquid scintillation counter (PerkinElmerLife Sciences).

Results

To investigate the effect of AngII on PAX2 expression in DU145 prostatecancer cells, PAX2 expression was examined following treatment withAngII over a 30 min to 48 hour period. As shown in FIG. 21, PAX2expression progressively increased over time following AngII treatment.Blocking RAS signaling by treating DU145 with Los significantly reducedPAX2 expression. Here, PAX2 expression was 37% after 48 hours and was50% after 72 hours of Los treatment compared to untreated control DU145cells (FIG. 22A). It is known that the AT2R receptor oppose the actionof the AT1R. Therefore, the effect of blocking the AT2R receptor on PAX2expression was examined. Treatment of DU145 with the AT2R blockerPD123319 resulted in a 7-fold increase in PAX2 expression after 48 hoursand an 8-fold increase after 96 hours of treatment (FIG. 22B).Collectively, these findings demonstrate that PAX2 expression isregulated by the ATR1 receptor.

It is known that AngII directly affects the proliferation of prostatecancer cells through AT1R-mediated activation of MAPK and STAT3phosphorylation. Treatment of DU145 with AngII resulted in a two- tothree-fold increase in proliferation rate (FIG. 23). However, treatmentwith Los decreased proliferated rates by 50%. In addition, blocking theAT1R receptor by pre-treating with Los for 30 min suppressed the effectof AngII on proliferation.

To further examine the role of the AT1R signaling in the regulation ofPAX2 expression and activation, the effect of blocking variouscomponents of the MAP kinase signaling pathway on PAX2 expression wasexamined. Here, DU145 cells treated with the MEK inhibitor U0126resulted in a significant reduction of PAX2 expression (FIG. 24).Furthermore, treatment with MEK/ERK inhibitor PD98059 also resulted indecreased PAX2. Treatment of DU145 cells with Los had no effect on ERKprotein levels, but reduced the amount of phospho-ERK (FIG. 25A).However, treatment of DU145 with Los resulted in a significant reductionof PAX2 expression. Similar results were observed with U0126 and PD98059(FIG. 25B). It is also known that PAX2 expression is regulated by STAT3which is a down-stream target of ERK. Treatment of DU145 with Los,U0126, and PD98059 reduced phospho-STAT3 protein levels (FIG. 25C).These results demonstrate that PAX2 is regulated via AT1R in prostatecancer cells.

In addition, the effect of AT1R signaling on PAX2 activation by JNK wasexamined Treatment of DU145 with Los, U0126, and PD98059 all resulted ina significant decrease or suppression of phospho-PAX2 protein levels(FIG. 26A). However, Los and U0126 did not decrease phospho-JNK proteinlevels (FIG. 26B). Therefore, the decrease in phospho-PAX2 appears to bedue to decreased PAX2 levels, but not decreased phosphorylation.

5-Aminoimidazole-4-carboxamide-1-β-4-ribofuranoside (AICAR) is widelyused as an AMP-kinase activator, which regulates energy homeostasis andresponse to metabolic stress. Recent reports have indicatedanti-proliferative and pro-apoptotic action of activated AMPK usingpharmacological agents or AMPK overexpression. AMPK activation has beenshown to induce apoptosis in human gastric cancer cells, lung cancercells, prostate cancer, pancreatic cells, and hepatic carcinoma cellsand enhance oxidative stress induced apoptosis in mouse neuroblastomacells, by various mechanisms that include inhibition of fatty acidsynthase pathway and induction of stress kinases and caspase 3. Inaddition, treatment of PC3 prostate cancer cells increased expression ofp21, p27, and p53 proteins and inhibition of PI3K-Akt pathway. All ofthese pathways are directly or indirectly regulated by PAX2. Treatmentof prostate cancer cells with AICAR resulted in the suppression of PAX2pression expression (FIG. 25B) as well as its activated formphosphor-PAX2 (FIG. 26A). In addition, phospho-STAT3 which regulatedPAX2 expression was also suppressed (FIG. 25C).

Finally, it was hypothesized that aberrant RAS signaling which leads toupregulation and overexpression of PAX2 suppresses the expression of theDEFB1 tumor suppressor gene. To investigate this, the normal prostateepithelial primary culture hPrEC was treated with AngII and examinedboth PAX2 and DEFB1 expression levels. An inverse relationship betweenDEFB1 and PAX2 expression was discovered in normal prostate cells versusprostate cancer cells. As shown in FIG. 27, untreated hPrEC exhibited10% relative PAX2 expression compared to expression in PC3 prostatecancer cells. Conversely, untreated PAX2 exhibited only 2% relativeDEFB1 expression compared to expression in hPrEC. Following 72 hours oftreatment with 10 uM of AngII, there was a 35% decrease in DEFB1expression compared to untreated hPrEC, and by 96 hours there was a 50%decrease in DEFB1 expression compared to untreated hPrEC cells. However,there was 66% increase in PAX2 expression at 72 hours, and by 96 hoursthere was a 79% increase in PAX2 expression compared to untreated hPrECcells. Furthermore, the increase in PAX2 expression in hPrEC after 72hours was 77% of PAX2 levels observed in PC3 prostate cancer cells.After 96 hours of AngII treatment PAX2 expression was 89% of PAX2expression in PC3. These results demonstrate that deregulated RASsignaling suppresses DEFB1 expression via the upregulation of PAX2expression in prostate cells.

Inhibition of apoptosis is a critical pathophysiological factor thatcontributes to the development of cancer. Despite significant advancesin cancer therapeutics, little progress has been made in the treatmentof advanced disease. Given that carcinogenesis is a multiyear,multistep, multipath disease of progression, chemoprevention through theuse of drug or other agents to inhibit, delay, or reverse this processhas been recognized as a very promising area of cancer research.Successful drug treatment for the chemoprevention of prostate cancerrequires the use of therapeutics with specific effects on target cellswhile maintaining minimal clinical effects on the host with the overallgoal of suppressing cancer development. Therefore, understanding themechanisms in early stage carcinogenesis is critical in determining theefficacy of a specific treatment. The significance of aberrant PAX2expression and its abrogation of apoptosis, with subsequent contributionto tumor formation, suggest that it may be a suitable target forprostate cancer treatment. PAX2 was regulated by the AT1R in prostatecancer (FIG. 28). In this, deregulated RAS signaling resulted inincreased PAX2 oncogene expression, and a decrease in the expression ofDEFB1 tumor suppressor. Therefore, the use of AT1R antagonists decreasesPAX2 expression and results in increased prostate cancer cell death viare-expression of DEFB1 (FIG. 29). These results offer a novel findingthat targeting PAX2 expression via the Renin-Angiotensin signalingpathway, the AMP Kinase pathway, or other methods involving theinactivation of the PAX2 protein (i.e. anti-PAX2 antibody vaccination)may be a viable target for cancer prevention (Table 4).

TABLE 4 Compounds Utilized to Inhibit PAX2 Expression forChemoprevention NAME Drug Class Drug 1 Losartan Angiotensin Type 1Receptor blocker Drug 2 PD123319 Angiotensin Type 2 Receptor blockerDrug 3 U0126 MEK inhibitor Drug 4 PD98059 MEK/ERK inhibitor Drug 5 AICARAMP kinase inducer Target Drug Function Drug A Anti-PAX2 Antibody PAX2Vaccine Drug B Angiotensinogen Renin-AngII pathway inhibitor Drug CAngiotensin Renin-AngII pathway inhibitor Converting Enzyme

This study demonstrates that the upregulation of the PAX2 oncogene inprostate cancer is due to deregulated RAS signaling. PAX2 expression isregulated by the ERK 1/2 signaling pathway which is mediated by theAngiotensin type I receptor. In addition, blocking the AT1R withLosartan (Los) suppresses PAX2 expression. In addition, AICAR which isan AMPK activator has also shown promise as a potential PAX2 inhibitor.Collectively, these studies strongly implicate these classes of drugs aspotential suppressors of PAX2 expression and may ultimately serve asnovels chemoprevention agents.

Example 9: PAX2-DEFB1 Expression Level as a Grading Tool for ProstateTissue and Predictor of Prostate Cancer Development Materials andMethods

QRT-PCR Analysis: Prostate sections were collected from patients thatunderwent radical prostatectomies. Following pathological examination,laser capture microdisection was performed to isolate areas of Normal,Proliferative Intraepithelial Neoplasia (PIN) and Cancerous tissue.QRT-PCR was performed as previously described to assess expression. DEFBland PAX2 expression in each region and GAPDH was used as an internalcontrol.

Blood collection and RNA isolation: For QRT-PCR, blood (2.5 ml) fromeach individual was colleted into a PAXgene™ Blood RNA tube (QIAGEN)following the manufacturer's protocol. Whole blood was thoroughly mixedwith PAXgene stabilization reagent and stored at room temperature for 6hours prior to RNA extraction. Total RNA was then extracted using thePAXgene™ Blood RNA kit according to the manufacturer's directions(QIAGEN). In order to remove contaminating genomic DNA, total RNAsamples absorbed to the PAXgene™ Blood RNA System spin column wasincubated with DNase I (QIAGEN) at 25° C. for 20 min to remove genomicDNA. Total RNA was eluted, quantitated, and QRT-PCR is performed aspreviously mentioned to compare PAX2 and DEFB1 expression ratios.

Results

QRT-PCR analysis of LCM normal tissue demonstrated that patients withrelative DEFB1 expression levels greater than 0.005 have a lower GleasonScore compared to those with expression levels lower than 0.005 (FIG.30). Thus, there is an inverse relationship between DEFB1 expression andGleason score. Conversely, there was a positive correlation between PAX2expression and Gleason score in malignant prostate tissue and PIN (FIG.30).

The PAX2 and DEFB1 expression levels in normal, PIN and canceroustissues from separate patients were calculated and compared (FIGS. 31Aand 31B). Overall, PAX2 expression levels relative to GAPDH internalcontrol ranged between 0 and 0.2 in normal (benign) tissue, 0.2 and 0.3in PIN, and between 0.3 and 0.5 in cancerous (malignant) tissue (FIG.32). For DEFB1 there was an inverse relationship compared to PAX2. Here,DEFB1 expression levels relative to GAPDH internal control rangedbetween 0.06 and 0.005 in normal (benign) tissue, 0.005 and 0.003 inPIN, and between 0.003 and 0.001 in cancerous (malignant) tissue.Therefore, disclosed is a predictive scale, designated as DonldPredictive Factor (DPF), which utilizes the PAX2-DEFB1 expression ratioas a prognosticator of benign, precancerous (PIN) and malignant prostatetissue. Tissues with PAX2-DEFB1 ratios between 0 and 39 based on the DPFwill represent normal (pathologically benign). Tissue with a PAX2-DEFB1ratio between 40 and 99 will represent PIN (pre-cancerous) based on theDPF scale. Finally, tissue with a PAX2-DEFB1 ratio between 100 and 500will be malignant (low to high grade cancer).

There currently is a critical need for predictive biomarkers forprostate cancer development. It is known that the onset of prostatecancer occurs long before the disease is detectable by current screeningmethods such as the PSA test or the digital rectal exam. It is thoughtthat a reliable test which could monitor the progression and early onsetof prostate cancer would greatly reduce the mortality rate through moreeffective disease management. Disclosed herein is a predictive index toallow physicians to know well in advance the pathological state of theprostate. The DPF measures the decrease in the PAX2-DEFB1 expressionratio associated with prostate disease progression. This powerfulmeasure can not only predict the likelihood of a patient developingprostate cancer, but also may pinpoint the early onset of pre-malignantcancer. Ultimately, this tool can allow physicians to segregate whichpatients have more aggressive disease from those which do not.

The identification of cancer-specific markers has been utilized to helpidentify circulating tumor cells (CTCs). There is also emerging evidencewhich demonstrates that detection of tumor cells disseminated inperipheral blood can provide clinically important data for tumorstaging, prognostication, and identification of surrogate markers forearly assessment of the effectiveness of adjuvant therapy. Furthermore,by comparing gene expression profiling of all circulating cells, one canexamine the expression of the DEFB1 and PAX2 genes which play a role in“immunosurveillance” and “cancer survival”, respectively as aprognosticator for the early detection of prostate cancer.

Example 10: Functional Analysis of the Host Defense Peptide Human BetaDefensin-1: New Insight into its Potential Role in Cancer Materials andMethods

Cell culture: The prostate cancer cell lines were cultured as describedin Example 1. The hPrEC primary culture was obtained from Cambrex BioScience, Inc. (Walkersville, Md.) and cells were grown in prostateepithelium basal media.

Tissue samples and laser capture microdissection: Prostate tissues wereobtained from patients who provided informed consent prior to undergoingradical prostatectomy. Samples were acquired through the Hollings CancerCenter tumor bank in accordance with an Institutional ReviewBoard-approved protocol. This included guidelines for the processing,sectioning, histological characterization, RNA purification and PCRamplification of samples. Prostate specimens received from the surgeonsand pathologists were immediately frozen in OCT compound. Each OCT blockwas cut to produce serial sections which were stained and examined.Areas containing benign cells, prostatic intraepithelial neoplasia(PIN), and cancer were identified and used to guide our selection ofregions from unstained slides using the Arcturus PixCell II System(Sunnyvale, Calif.). Caps containing captured material were exposed to20 μl of lysate from the Arcturus Pico Pure RNA Isolation Kit andprocessed immediately. RNA quantity and quality was evaluated using setsof primers that produce 5′ amplicons. The sets include those for theribosomal protein L32 (the 3′ amplicon and the 5′ amplicon are 298 basesapart), for the glucose phosphate isomerase (391 bases apart), and forthe glucose phosphate isomerase (842 bases apart). Ratios of 0.95 to0.80 were routinely obtained for these primer sets using samples from avariety of prepared tissues. Additional tumor and normal samples weregrossly dissected by pathologists, snap frozen in liquid nitrogen andevaluated for hBD-1 and cMYC expression.

Cloning of hBD-1 gene: hBD-1 cDNA was generated from RNA by reversetranscription-PCR using primers generated from the published hBD-1sequence (accession no. U50930) (Ganz, 2004). The PCR primers weredesigned to contain ClaI and KpnI restriction sites. hBD-1 PCR productswere restriction digested with ClaI and KpnI and ligated into a TAcloning vector. The TA/hBD1 vector was then transfected into the XL-1Blue strain of E. coli by heat shock and individual clones were selectedand expanded. Plasmids were isolated by Cell Culture DNA Midiprep(Qiagen, Valencia, Calif.) and sequence integrity verified by automatedsequencing. The hBD-1 gene fragment was then ligated into the pTRE2digested with ClaI and KpnI, which served as an intermediate vector fororientation purposes. The pTRE2/hBD-1 construct was digested with ApaIand KpnI to excise the hBD-1 insert. The insert was ligated into pINDvector of the Ecdysone Inducible Expression System (Invitrogen,Carlsbad, Calif.) also double digested with ApaI and KpnI. The constructwas transfected into E. coli and individual clones were selected andexpanded. Plasmids were isolated and sequence integrity of pIND/hBD-1was again verified by automated sequencing.

Transfection: Cells (1×10⁶) were seeded onto 100-mm Petri dishes andgrown overnight. Next, the cells were co-transfected using Lipofectamine2000 (Invitrogen) with 1 pg of pvgRXR plasmid, which expresses theheterodimeric ecdysone receptor, and 1 μg of the pIND/hBD-1 vectorconstruct or pIND/β-galactosidase (β-gal) control vector in Opti-MEMmedia (Life Technologies, Inc.). Transfection efficiency was determinedby inducing β-gal expression with Ponasterone A (PonA) and stainingcells with a β-galactosidase detection kit (Invitrogen). Assessment oftransfection efficiency by counting positive staining (blue) colonieswhich demonstrated that 60-85% of cells expressed β-galactosidase forthe cell lines.

Immunocytochemistry: In order to verify hBD-1 protein expression, DU145and hPrEC cells were seeded onto 2-chamber culture slides (BD Falcon,USA) at 1.5-2×10⁴ cells per chamber. DU145 cells transfected with pvgRXRalone (control) or with the hBD-1 plasmid were induced for 18 h withmedia containing 10 μM Pon A, while untransfected cells received freshgrowth media. Following induction, cells were washed in 1×PBS and fixedfor 1 h at room temperature with 4% paraformaldehyde. Cells were thenwashed six times with 1×PBS and blocked in 1×PBS supplemented with 2%BSA, 0.8% normal goat serum (Vector Laboratories, Inc., Burlingame,Calif.) and 0.4% Triton-X 100 for 1 h at room temperature. Next, cellswere incubated overnight in primary rabbit anti-human BD-1 polyclonalantibody (PeproTech Inc., Rocky Hill, N.J.) diluted 1:1000 in blockingsolution. Following this, cells were washed six times with blockingsolution and incubated for 1 h at room temperature in Alexa Fluor 488goat anti-rabbit IgG (H+L) secondary antibody at a dilution of 1:1000 inblocking solution. After washing cells with blocking solution six times,coverslips were mounted with Gel Mount (Biomeda, Foster City, Calif.).Finally, cells were viewed under differential interference contrast(DIC) and under laser excitation at 488 nm. The fluorescent signal wasanalyzed by confocal microscopy (Zeiss LSM 5 Pascal) using a 63×DIC oillens with a Vario 2 RGB Laser Scanning Module. The digital images wereexported into Photoshop CS Software (Adobe Systems) for image processingand hard copy presentation.

RNA isolation and quantitative RT-PCR: QRT-PCR was performed aspreviously described (Gibson et al., 2007). Briefly, total RNA (0.5 μgper reaction) from tissue sections were reverse transcribed into cDNAutilizing random primers (Promega). Two-step QRT-PCR was performed oncDNA generated using the MultiScribe Reverse Transcriptase from theTaqMan Reverse Transcription System and the SYBR Green PCR Master Mix(Applied Biosystems, Foster City, Calif.). The primer pairs for hBD-1and c-MYC were generated from the published sequences (Table 5). Fortycycles of PCR were performed under standard conditions using anannealing temperature of 56.4° C. for hBD-1 and c-MYC and 55° C. forPAX2. In addition, β-actin (Table 5) was amplified as a housekeepinggene to normalize the initial content of total cDNA. Gene expression inbenign prostate tissue samples was calculated as the expression ratiocompared to β-actin. Levels of hBD-1 expression in malignant prostatetissue, hPREC prostate primary culture, and prostate cancer cell linesbefore and after induction were calculated relative to the average levelof hBD-1 expression in hPrEC cells. As a negative control, QRT-PCRreactions without cDNA template were also performed. All reactions wererun a minimum of three times.

TABLE 5 Sequences of QRT-PCR primers Sense (5′-3′) Antisense (5′-3′)β-Actin CCTGGCACCCAGCACAAT GCCGATCCACACGGAGTA (SEQ ID NO: 51)CT (SEQ ID NO: 52) hBD-1 TCAGCAGTGGAGGGCAATG CCTCTGTAACAGGTGCCTT(SEQ ID NO: 65) GAAT (SEQ ID NO: 66) cMYC ACAGCAAACCTCCTCACAGTGGAGACGTGGCACCTCTTG CC (SEQ ID NO: 67) (SEQ ID NO: 68)

MTT cell viability assay: To examine the effects of hBD-1 on cellgrowth, metabolic 3-[4,5-dimethylthiazol-2yl]-2,5-diphenyl tetrazoliumbromide (MTT) assay was performed. DU145, LNCaP, PC3 and PC3/AR+ cellsco-transfected with pvgRXR plasmid and pIND/hBD-1 construct or controlpvgRXR plasmid were seeded onto a 96-well plate at 1-5×10³ cells perwell. Twenty-four hours after seeding, fresh growth medium was addedcontaining 10 μM Pon A daily to induce hBD-1 expression for 24, 48 and72 h after which the MTT assay was performed according to themanufacturer's instructions (Promega). Reactions were performed threetimes in triplicate.

Analysis of membrane integrity: Acridine orange (AO)/ethidium bromide(EtBr) dual staining was performed to identify changes in cell membraneintegrity, as well as apoptotic cells by staining the condensedchromatin. AO stains viable cells and early apoptotic cells, whereasEtBr stains late stage apoptotic cells that have compromised membranes.Briefly, PC3, DU145 and LNCaP cells were seeded into 2-chamber cultureslides (BD Falcon). Cells transfected with empty plasmid or hBD-1plasmid were induced for 24 or 48 h with media containing 10 μM Pon A,while control cells received fresh growth media at each time point.After induction, cells were washed once with PBS and stained with 2 mlof a mixture (1:1) of AO (Sigma, St. Louis, Mo.) and EtBr (Promega) (5μg/ml) solution for 5 min and were again washed with PBS.

Fluorescence was viewed by a Zeiss LSM 5 Pascal Vario 2 Laser ScanningConfocal Microscope (Carl Zeiss). The excitation color wheel containsBS505-530 (green) and LP560 (red) filter blocks which allowed for theseparation of emitted green light from AO into the green channel and redlight from EtBr into the red channel. The laser power output and gaincontrol settings within each individual experiment were identicalbetween control and hBD-1 induced cells. The excitation was provided bya Kr/Ar mixed gas laser at wavelengths of 543 nm for AO and 488 nm forEtBr. Slides were analyzed under 40× magnification and digital imageswere stored as uncompressed TIFF files and exported into Photoshop CSsoftware (Adobe Systems) for image processing and hard copypresentation.

Flow cytometry: PC3 and DU145 cells transfected with the hBD-1expression system were grown in 60-mm dishes and induced for 12, 24, and48 h with 10 μM Pon A. The cells were harvested and analyzed by flowcytometry as described in Example 1.

Caspase detection: Detection of caspase activity in the prostate cancercell lines was performed described in Example 1.

siRNA silencing of PAX2: SiRNA knock-down and verification was performedas described in Example 2.

Results

hBD-1 expression in prostate tissue: 82% of prostate cancer frozentissue sections analyzed exhibited little or no expression of hBD-1(Donald et al., 2003). To compare hBD-1 expression levels, QRTPCRanalysis was performed on normal prostate tissue obtained by grossdissection or LCM of normal prostate tissue adjacent to malignantregions which were randomly chosen. Here, hBD-1 was detected in all ofthe gross dissected normal clinical samples with a range of expressionthat represents approximately a 6.6-fold difference in expression levels(FIG. 33A). LCM captured normal tissue samples expressed hBD-1 at levelsin a range that represents a 32-fold difference in expression (FIG.33B). Matching sample numbers to corresponding patient profiles revealedthat in most cases, the hBD-1 expression level was higher in patientsamples with a Gleason score of 6 than in patient samples with a Gleasonscore of 7. In addition, a comparison of hBD-1 expression levels intissue obtained by gross dissection and LCM from the same patient,#1343, demonstrated an 854-fold difference in expression between the twoisolation techniques. Therefore, these results indicate that LCMprovides a more sensitive technique to assess hBD-1 expression inprostate tissue.

hBD-1 expression in prostate cell lines: To verify upregulation of hBD-1in the prostate cancer cell lines after transfection with the hBD-1expression system, QRTPCR was performed. In addition, no templatenegative controls were also performed, and amplification products wereverified by gel electrophoresis. Here, hBD-1 expression wassignificantly lower in the prostate cancer cell lines compared to hPrECcells. Following a 24 h induction period, relative expression levels ofhBD-1 significantly increased in DU145, PC3 and LNCaP as compared to thecell lines prior to hBD-1 induction (FIG. 34A).

Next, protein expression of hBD-1 in was verified DU145 cellstransfected with the hBD-1 expression system after induction with Pon Aby immunocytochemistry. As a positive control, hBD-1 expressing hPrECprostate epithelial cells were also examined. Cells were stained withprimary antibody against hBD-1 and protein expression was monitoredbased on the green fluorescence of the secondary antibody (FIG. 34B).Analysis of cells under DIC verify the presence of hPrEC cells and DU145cells induced for hBD-1 expression at 18 h. Excitation by the confocallaser at 488 nm produced revealed green fluorescence indicating thepresence of hBD-1 protein in hPrEC as a positive control. However, therewas no detectable green fluorescence in control DU145 cells and emptyplasmid induced DU145 cells demonstrating no hBD-1 expression. Confocalanalysis of DU145 cells induced for hBD-1 expression revealed greenfluorescence indicating the presence of hBD-1 protein followinginduction with Pon A.

Expression of hBD-1 results in decreased cell viability: MTT assay wasperformed to assess the effect of hBD-1 expression on relative cellviability in DU145, PC3, PC3/AR+ and LNCaP prostate cancer cell lines.MTT analysis with empty vector exhibited no statistical significantchange in cell viability. Twenty-four hours following hBD-1 induction,relative cell viability was 72% in DU145 and 56% in PC3 cells, and after48 h cell viability was reduced to 49% in DU145 and 37% in PC3 cells(FIG. 35). Following 72 h of hBD-1 induction, relative cell viabilitydecreased further to 44% in DU145 and 29% PC3 cells. Conversely, therewas no significant effect on the viability of LNCaP cells. In order toassess whether the resistance to hBD-1 cytotoxicity observed in LNCaPwas due to the presence of the androgen receptor (AR), the hBD-1cytotoxicity in PC3 cells was examined with ectopic AR expression(PC3/AR+). Here, there was no difference between PC3/AR+ and PC3 cells.Therefore, the data indicates that that hBD-1 is cytotoxic specificallyto late-stage prostate cancer cells.

In order to determine whether the effects of hBD-1 on PC3 and DU145 werecytostatic or cytotoxic, FACS analysis was performed to measure celldeath. Under normal growth conditions, more than 90% of PC3 and DU145cultures were viable and non-apoptotic (lower left quadrant) and did notstain with annexin V or PI. After inducing hBD-1 expression in PC3cells, the number of cells undergoing early apoptosis and lateapoptosis/necrosis (lower and upper right quadrants, respectively)totaled 10% at 12 h, 20% at 24 h, and 44% at 48 h (FIG. 4B). For DU145cells, the number of cells undergoing early apoptosis and lateapoptosis/necrosis totaled 12% after 12 h, 34% at 24 h, and 59% after 48h of induction (FIG. 4A). No increase in apoptosis was observed in cellscontaining empty plasmid following induction with Pon A. Annexin V andpropidium iodide uptake studies have demonstrated that hBD-1 hascytotoxic activity against DU145 and PC3 prostate cancer cells andresults indicate apoptosis as a mechanism of cell death.

hBD-1 causes alterations in membrane integrity and caspase activation:It was investigated whether the cell death observed in prostate cancercells after hBD-1 induction is caspase-mediated apoptosis. To betterunderstand the cellular mechanisms involved in hBD-1 expression,confocal laser microscopic analysis was performed (FIGS. 5A-5L) on DU145and LNCaP cells induced for hBD-1 expression. Pan-caspase activation wasmonitored based on the binding and cleavage of green fluorescingFAM-VAD-FMK to caspases in cells actively undergoing apoptosis. Analysisof cells under DIC showed the presence of viable control DU145 (FIG. 5A)and LNCaP (FIG. 5E) cells at 0 h. Excitation by the confocal laser at488 nm produced no detectable green staining which indicates no caspaseactivity in DU145 (FIG. 5B) or LNCaP (FIG. 5F) control cells. Followinginduction for 24 h, DU145 (FIG. 5C) and LNCaP (FIG. 5G) cells were againvisible under DIC. Confocal analysis under fluorescence revealed greenstaining in DU145 (FIG. 5D) cells indicating pan-caspase activity afterthe induction of hBD-1 expression. However, there was no green stainingin LNCaP (FIG. 5H) cells induced for hBD-1 expression. Therefore, celldeath observed following induction of hBD-1 is caspase-mediatedapoptosis.

The proposed mechanism of antimicrobial activity of defensin peptides isthe disruption of the microbial membrane due to pore formation (Papo andShai, 2005). In order to determine if hBD-1 expression altered membraneintegrity EtBr uptake was examined by confocal analysis. Intact cellswere stained green due to AO which is membrane permeable, while onlycells with compromised plasma membranes stained red due to incorporationof membrane impermeable EtBr. Control DU145 and PC3 cells stainedpositively with AO and emitted green color, but did not stain with EtBr.However, hBD-1 induction in both DU145 and PC3 resulted in theaccumulation of EtBr in the cytoplasm at 24 as indicated by the redstaining. By 48 h, DU145 and PC3 possessed condensed nuclei and appearedyellow due to the colocalization of green and red staining from AO andEtBr, respectively. Conversely, there were no observable alterations tomembrane integrity in LNCaP cells after 48 h of induction as indicatedby positive green fluorescence with AO, but lack of red EtBrfluorescence. This finding indicates that alterations to membraneintegrity and permeabiization in response to hBD-1 expression differbetween early- and late-stage prostate cancer cells.

Comparison of hBD-1 and cMYC expression levels: QRT-PCR analysis wasperformed on LCM prostate tissue sections from three patients (FIG. 34).In patient #1457, hBD-1 expression exhibited a 2.7-fold decrease fromnormal to PIN, a 3.5-fold decrease from PIN to tumor and a 9.3-folddecrease from normal to tumor (FIG. 36A). Likewise, cMYC expressionfollowed a similar expression pattern in patient #1457 where expressiondecreased by 1.7-fold from normal to PIN, 1.7-fold from PIN to tumor and2.8-fold from normal to tumor (FIG. 36B). In addition, there was astatistically significant decrease in cMYC expression in the other twopatients. Patient #1569 had a 2.3-fold decrease from normal to PIN,while in patient #1586 there was a 1.8-fold decrease from normal to PIN,a 4.3-fold decrease from PIN to tumor and a 7.9-fold decrease fromnormal to tumor.

Induction of hBD-1 expression following PAX2 inhibition: To furtherexamine the role of PAX2 in regulating hBD-1 expression, siRNA wasutilized to knockdown PAX2 expression and QRT-PCR performed to monitorhBD-1 expression. Treatment of hPrEC cells with PAX2 siRNA exhibited noeffect on hBD-1 expression (FIG. 37). However, PAX2 knockdown resultedin a 42-fold increase in LNCaP, a 37-fold increase in PC3 and a1026-fold increase in DU145 expression of hBD-1 compared to untreatedcells. As a negative control, cells were treated with non-specific siRNAwhich had no significant effect on hBD-1 expression.

Example 11: Inhibition of PAX2 Expression Results in Alternate CellDeath Pathways in Prostate Cancer Cells Differing in P53 StatusMaterials and Methods

Cell lines: The cancer cell lines PC3, DU145 and LNCaP, which all differin p53 mutational status (Table 6), were cultured as described inExample 1. The prostate epithelial cell line HPrEC was obtained fromCambrex Bio Science, Inc., (Walkersville, Md.) and were cultured inprostate epithelium basal media. Cells were maintained at 37° C. in 5%CO2.

TABLE 6 p53 gene mutation in prostate cancer cell lines Nucleotide Aminoacid change change Gene status Reference CCT-CTT Pro-LeuGain/loss-of-function Tepper et al. 2005; Bodhoven et al. 2003 GTT-TTTVal-Phe Deleted a C, Frame-shift No activity Isaacs et al. 1991 GCC-GCNo deletion, — Normal Carroll et al. 1993 wild-type function

siRNA silencing of PAX2: siRNA silencing of PAX2 was performed sdescribed in Example 2.

Western analysis: Western blot was performed as described in Example 2.Blots were then probed with rabbit anti-PAX2 primary antibody (Zymed,San Francisco, Calif.) at a 1:1000 dilution. After washing, themembranes were incubated with anti-rabbit antibody conjugated tohorseradish peroxidase (HRP) (dilution 1:5000; Sigma), and signaldetection was visualized using chemiluminescence reagents (Pierce) on anAlpha Innotech Fluorchem 8900. As a control, blots were stripped andreprobed with mouse anti-β-actin primary antibody (1:5000;Sigma-Aldrich) and HRP-conjugated anti-mouse secondary antibody (1:5000;Sigma-Aldrich), and signal detection was again visualized.

Phase contrast microscopy: The effect of PAX2 knockdown on cell numberwas analyzed by phase contrast microscopy as described in Example 1.

MTT cytotoxicity assay: MTT cytotoxicity assay was performed asdescribed in Example 1.

Pan-caspase detection: Detection of caspase activity in the prostatecancer cell lines was performed as described in Example 1.

Quantitative real-time RT-PCR: To verify changes in gene expressionfollowing PAX2 knockdown in PC3, DU145 and LNCaP cell lines,quantitative real-time RT-PCR was performed as described in Example 1.The primer pairs for BAX, BID, BCL-2, AKT and BAD were generated fromthe published sequences (Table 7). Reactions were performed in MicroAmpOptical 96-well Reaction Plate (PE Biosystems). Forty cycles of PCR wereperformed under standard conditions using an annealing temperature of60° C. Quantification was determined by the cycle number whereexponential amplification began (threshold value) and averaged from thevalues obtained from the triplicate repeats. There was an inverserelationship between message level and threshold value. In addition,GAPDH was used as a housekeeping gene to normalize the initial contentof total cDNA. Relative expression was calculated as the ratio betweeneach genes and GAPDH. All reactions were carried out in triplicate.

TABLE 10 Quantitative RT-PCR primers Sense (5′-3′) Antisense (5′-3′)GAPDH CCACCCATGGCAAATTCCAT TCTAGACGGCAGGTCAGGTC GGCA (SEQ ID NO: 55)AACC (SEQ ID NO: 56) BAD CTCAGGCCTATGCAAAAAGA GCCCTCCCTCCAAAGGAGACGGA (SEQ ID NO: 57) (SEQ ID NO: 58) BID AACCTACGCACCTACGTGAGCGTTCAGTCCATCCCATTTC GAG (SEQ ID NO: 59) TG (SEQ ID NO: 60) BAXGACACCTGAGCTGACCTTGG GAGGAAGTCCAGTGTCCAGC (SEQ ID NO: 61)(SEQ ID NO: 62) BCL-2 TATGATACCCGGGAGATCGT GTGCAGATGCCGGTTCAGGTGATC (SEQ ID NO: 69) ACTC (SEQ ID NO: 70) AKT TCAGCCCTGGACTACCTGCAGAGGTCCCGGTACACCACGT (SEQ ID NO: 71) (SEQ ID NO: 72)

Membrane permeability assay: Membrane permeability assay was performed sdescribed in Example 3.

Results

Analysis of PAX2 protein expression in prostate cells: PAX2 proteinexpression was examined by Western analysis in HPrEC prostate primaryculture and in LNCaP, DU145 and PC3 prostate cancer cell lines. Here,PAX2 protein was detected in all of the prostate cancer cell lines (FIG.38A). However, no PAX2 protein was detectable in HPrEC. Blots werestripped and re-probed for β-actin as internal control to ensure equalloading. PAX2 protein expression was also monitored after selectivetargeting and inhibition by PAX2 specific siRNA in DU145, PC3 and LNCaPprostate cancer cell lines. Cells were given a single round oftransfection with the pool of PAX2 siRNA over a 6-day treatment period.PAX2 protein was expressed in control cells treated with media only.Specific targeting of PAX2 mRNA was confirmed by observing knockdown ofPAX2 protein in all three cell lines (FIG. 38B).

Effect of PAX2 knockdown on prostate cancer cell growth: The effect ofPAX2 siRNA on cell number and cell viability was analyzed using lightmicroscopy and MTT analysis. To examine the effect of PAX2 siRNA on cellnumber, PC3, DU145 and LNCaP cell lines were transfected with mediaonly, non-specific siRNA or PAX2 siRNA over a period of 6 days. Each ofthe cell lines reached a confluency of 80-90% in 60 mm culture dishescontaining media only. Treatment of HPrEC, DU145, PC3 and LNCaP cellswith non-specific siRNA appeared to have little to no effect on cellgrowth compared to cell treated with media only (FIGS. 39A, C, and E,respectively). Treatment of the PAX2-null cell line HPrEC with PAX2siRNA appeared to have no significant effect on cell growth (FIG. 39B).However, treatment of the prostate cancer cell lines DU145, PC3 andLNCaP with PAX2 siRNA resulted in a significant decrease in cell number(FIGS. 39D, F, and H, respectively).

Effect of PAX2 knockdown on prostate cancer cell viability: Cellviability was measured after 2-, 4-, and 6-day exposure times. Percentviability was calculated as the ratio of the 570-630 nm absorbance ofcell treated with PAX2 siRNA divided by untreated control cells. Asnegative controls, cell viability was measured after each treatmentperiod with negative control non-specific siRNA or transfection withreagent alone. Relative cell viability was calculated by dividingpercent viability following PAX2 siRNA treatment by percent viabilityfollowing treatment with non-specific siRNA (FIG. 40). After 2 days oftreatment, relative viability was 116% in DU145, 81% in PC3 and 98% inLNCaP. After 4 days of treatment, relative cell viability decreased to69% in DU145, 79% in PC3, and 80% in LNCaP. Finally, by 6 days relativeviability was 63% in DU145, 43% in PC3 and 44% in LNCaP. In addition,cell viability was also measured following treatment with transfectionreagent alone. Here, each cell line exhibited no significant decrease incell viability.

Detection of pan-caspase activity: Caspase activity was detected byconfocal laser microscopic analysis. LNCaP, DU145 and PC3 cells weretreated with PAX2 siRNA and activity was monitored based on the bindingof FAM-labeled peptide to caspases in cells actively undergoingapoptosis which will fluoresce green. Analysis of cells with media onlyshows the presence of viable LNCaP, DU145 and PC3 cells, respectively.Excitation by the confocal laser at 488 nm produced no detectable greenstaining which indicates no caspase activity in the untreated cells(FIGS. 41A, C, and E, respectively). Following 4 days of treatment withPAX2 siRNA, LNCaP, DU145 and PC3 cells under fluorescence presentedgreen staining indicating caspase activity (FIGS. 41B, D, and F,respectively).

Effect of PAX2 inhibition on apoptotic factors: LNCaP, DU145 and PC3cells were treated with siRNA against PAX2 for 4 days and expression ofboth pro- and anti-apoptotic factors were measured by QRTPCR. FollowingPAX2 knockdown, analysis of BAD revealed a 2-fold in LNCaP, 1.58-fold inDU145 and 1.375 in PC3 (FIG. 42A). Expression levels of BID increased by1.38-fold in LNCaP and a 1.78-fold increase in DU145, but there was nostatistically significant difference in BID observed in PC3 aftersuppressing PAX2 expression (FIG. 42B). Analysis of the anti-apoptoticfactor AKT revealed a 1.25-fold decrease in expression in LNCaP and a1.28-fold decrease in DU145 following treatment, but no change wasobserved in PC3 (FIG. 42C).

Analysis of membrane integrity and necrosis: Membrane integrity wasmonitored by confocal analysis in LNCaP, DU145 and PC3 cells. Here,intact cells stained green due to AO which is membrane permeable, whilewith compromised plasma membranes would stained red due to incorporationof membrane impermeable EtBr into the cytoplasm, and yellow due toco-localization of AO and EtBr in the nuclei. Untreated LNCaP, DU145 andPC3 cells stained positively with AO and emitted green color, but didnot stain with EtBr. Following PAX2 knockdown, there were no observablealterations to membrane integrity in LNCaP cells as indicated bypositive green fluorescence with AO and absence of red EtBrfluorescence. These finding further indicate that LNCaP cells can beundergoing apoptotic, but not necrotic cell death following PAX2knockdown. Conversely, PAX2 knockdown in DU145 and PC3 resulted in theaccumulation of EtBr in the cytoplasm as indicated by the red staining.In addition, both DU145 and PC3 possessed condensed nuclei whichappeared yellow due to the co-localization of green and red stainingfrom AO and EtBr, respectively. These results indicate that DU145 andPC3 are undergoing an alternate cell death pathway involving necroticcell death compared to LNCaP.

Example 12: PAX2 and DEFB-1 Expression in Breast Cancer Cell Lines andMammary Tissues with Ductal or Lobular Intraepithelial Neoplasia

PAX2 and DEFB-1 expression will be determined in breast biopsy samplesof ductal or lobular intraepithelial neoplasia, and in the followingbreast cancer cell lines:

BT-20: Isolated from a primary invasive ductal carcinoma; cell expressE-cadherin, ER, EGFR and uPA.

BT-474: Isolated from a primary invasive ductal carcinoma; cell expressE-cadherin, ER, PR, and have amplified HER2/neu.

Hs578 T: Isolated from a primary invasive ductal carcinoma; a cell linewas also established from normal adjacent tissue, termed Hs578Bst.

MCF-7: Established from a pleural effusion. The cells express ER and arethe most common example of estrogen-responsive breast cancer cells.

MDA-MB-231: Established from a pleural effusion. The cells areER-negative, E-cadherin negative and highly invasive in in vitro assays.

MDA-MB-361: Established from a brain metastasis. The cells express ER,PR, EGFR and HER2/neu.

MDA-MB-435: Established from a pleural effusion. The cells areER-negative, E-cadherin negative, and are highly invasive and metastaticin immunodeficient mice.

MDA-MB-468: Established from a pleural effusion. The cells haveamplified EGFR and are ER-negative.

SK-BR-3: Established from a pleural effusion. The cells have amplifiedHER/2neu, express EGFR and are ER-negative.

T-47D: established from a pleural effusion. The cells retain expressionof E-cadherin, ER and PR.

ZR-75-1: Established from ascites fluid. The cells express ER,E-cadherin, HER2/neu and VEGF.

The PAX2-to-DEFB-1 expression ratio will be determined using the methodsdescribed in Example 9.

Example 13: Expression of DEFB1 in Breast Cancer Cells

DEFB1 will be expressed in breast cancer cells using methods describedin Example 1. The cell viability and caspase activity will be determinedas described in Example 1.

Example 14: Inhibition of PAX2 Expression in Breast Cancer Cells

PAX2 expression in breast cancer cells will be inhibited using the siRNAdescribed in Example 2. The expression levels of pro-apoptotic genessuch as BAX, BID and BAD, the cell viability and caspase activity willbe determined as described in Example 2.

The anti-tumoral ability of DEFB1 will be evaluated by injecting breastcancer cells that overexpress DEFB1 into nude mice. Breast cancer cellswill be transfected with an expression vector carrying the DEFB1 gene.Cells expressing the exogenous DEFB1 gene will be selected and cloned.Only single-cell suspensions with a viability of >90% are used. Eachanimal receives approximately 500,000 cells administered subcutaneouslyinto the right flank of female nude mice. There are two groups, acontrol group injected with vector only clones and a group injected withthe DEFB1 over-expressing clones. 35 mice are in each group asdetermined by a statistician. Animals are weighed twice weekly, tumorgrowth monitored by calipers and tumor volumes determined using thefollowing formula: volume=0.5×(width)2×length. All animals aresacrificed by CO2 overdose when tumor size reaches 2 mm3 or 6 monthsfollowing implantation; tumors are excised, weighed and stored inneutral buffered formalin for pathological examination. Differences intumor growth between the groups are descriptively characterized throughsummary statistics and graphical displays. Statistical significance isevaluated with either the t-test or non-parametric equivalent.

Example 16: Effect of PAX2 siRNA on Tumor Growth In Vivo

Hairpin PAX2 siRNA template oligonucleotides utilized in the in vitrostudies are utilized to examine the effect of the up-regulation of DEFB1expression in vivo. The sense and antisense strand (see Table 3) areannealed and cloned into pSilencer 2.1 U6 hygro siRNA expression vector(Ambion) under the control of the human U6 RNA pol III promoter. Thecloned plasmid is sequenced, verified and transfected into breast cancercell lines. Scrambled shRNA is cloned and used as a negative control inthis study. Hygromycin resistant colonies are selected, cells areintroduced into the mice subcutaneously and tumor growth is monitored asdescribed above.

Example 17: Effect of Small Molecule Inhibitors of PAX2 Binding onBreast Cancer Cells

The alternative inhibitory oligonucleotides described in Example 6 willbe transfected into the breast cancer cells with lipofectamine reagentor Codebreaker transfection reagent (Promega, Inc). In order to confirmDNA-protein interactions, double stranded oligonucleotides will belabeled with [32P] dCTP and electrophoretic mobility shift assays areperformed DEFB1 expression will be monitored by QRT-PCR and Westernanalysis following treatment with oligonucleotides. Finally, cell deathwill be detected by MTT assay and flow cytometry as previouslydescribed.

The above description is for the purpose of teaching the person ofordinary skill in the art how to practice the present invention, and itis not intended to detail all those obvious modifications and variationsof it which will become apparent to the skilled worker upon reading thedescription. It is intended, however, that all such obviousmodifications and variations be included within the scope of the presentinvention, which is defined by the following claims. The claims areintended to cover the claimed components and steps in any sequence whichis effective to meet the objectives there intended, unless the contextspecifically indicates the contrary.

What is claimed is:
 1. A method of treating breast cancer in a subject, comprising: administering to the subject an anti-PAX2 composition, wherein the composition comprises an effective amount of an anti-PAX2 agent, wherein the anti-PAX2 agent is an antagonist of PAX2; and wherein the anti-PAX2 agent is not a STAT3 inhibitor.
 2. The method of claim 1, wherein the subject is estrogen receptor positive, progesterone receptor positive, or human epidermal growth factor 2 receptor positive.
 3. The method of claim 1, wherein the subject has been previously treated with an anti-estrogen receptor, anti-progesterone receptor, or an anti-human epidermal growth factor 2 receptor.
 4. The method of claim 1, further comprises the step of determining the PAX2-to-DEFB1 expression ratio in a breast cancer tissue from the subject.
 5. The method of claim 1, further comprises the step of determining the estrogen receptor/progesterone receptor/human epidermal growth factor 2 receptor status of said breast cancer tissue from said subject.
 6. The method of claim 1, wherein said anti-PAX2 agent is conjugated to a targeting moiety to target breast cancer tissue.
 7. The method of claim 6, wherein the targeting moiety is an antibody.
 8. The method of claim 6, wherein the targeting moiety is a receptor.
 9. The method of claim 6, wherein the targeting moiety is a receptor ligand.
 10. The method of claim 1, wherein the anti-PAX2 composition is administered intravenously.
 11. A method of treating breast cancer in a subject, comprising: administering to the subject an anti-PAX2 composition, wherein said composition comprises an effective amount of an anti-PAX2 agent, wherein the anti-PAX2 agent is an antagonist of PAX2; and wherein the subject is PAX2 positive.
 12. The method of claim 11, wherein the subject is estrogen receptor positive, progesterone receptor positive, or human epidermal growth factor 2 receptor positive.
 13. The method of claim 11, wherein the subject has been previously treated with an anti-estrogen receptor, anti-progesterone receptor, or an anti-human epidermal growth factor 2 receptor.
 14. The method of claim 11, further comprises the step of determining the PAX2-to-DEFB1 expression ratio in a breast cancer tissue from the subject.
 15. The method of claim 11, further comprises the step of determining the estrogen receptor/progesterone receptor/human epidermal growth factor 2 receptor status of said breast cancer tissue from said subject.
 16. The method of claim 11, wherein said anti-PAX2 agent is conjugated to a targeting moiety to target breast cancer tissue.
 17. The method of claim 16, wherein the targeting moiety is an antibody.
 18. The method of claim 16, wherein the targeting moiety is a receptor.
 19. The method of claim 16, wherein the targeting moiety is a receptor ligand.
 20. The method of claim 11, wherein the anti-PAX2 composition is administered intravenously. 